Aquisição de bactéria gram-negativa multidroga resistente ... · This thesis is in accordance with the following rules, validity at the moment of the publication. References: adapted

MARISTELA PINHEIRO FREIRE

Aquisição de bactéria gram-negativa

multidroga resistente antes do transplante

de fígado : o impacto no desfecho

Tese apresentada à Faculdade de Medicina da

Universidade de São Paulo para obtenção do título

de Doutora em Ciências

Programa de Doenças Infecciosas e Parasitárias

Orientador: Prof. Dr. Edson Abdala

São Paulo

2017

Dados Internacionais de Catalogação na Publicação (CIP)

Preparada pela Biblioteca da

Faculdade de Medicina da Universidade de São Paulo

reprodução autorizada pelo autor

Freire, Maristela Pinheiro

Aquisição de bactéria gram-negativa multidroga resistente antes do transplante

de fígado: o impacto no desfecho / Maristela Pinheiro Freire -- São Paulo, 2017.

Tese (doutorado)--Faculdade de Medicina da Universidade de São Paulo.

Programa de Doenças Infecciosas e Parasitárias.

Orientador: Edson Abdala.

Descritores: 1.Transplante de fígado 2.Bactéria multi-droga resistente

3.Fator de risco 4.Mortalidade 5.Bacilo Gram-negative 6.Resistência a

carbapênico

USP/FM/DBD-244/17

MARISTELA PINHEIRO FREIRE

Multidrug-resistant gram-negative bacteria

acquired before liver transplantation : the

impact on the outcome

Thesis presented at the Faculdade de Medicina da

Universidade de São Paulo for acquisition of the

title of Philosophiae Doctor (PhD)

Infectious Diseases Program

Advisor: Prof. Dr. Edson Abdala

São Paulo

2017

This thesis is in accordance with the following rules, validity at the moment of

the publication.

References: adapted from International Committee of Medical Journals Editors

(Vancouver).

Universidade de São Paulo. Faculdade de Medicina. Divisão de Biblioteca e

Documentação. Guia de apresentação de dissertações, teses e monografias.

Authors: Anneliese Carneiro da Cunha, Maria Julia de A. L. Freddi, Maria F.

Crestana, Marinalva de Souza Aragão, Suely Campos Cardoso, Valéria

Vilhena. 3a edition. São Paulo: Divisão de Biblioteca e Documentação: 2011.

Abbreviations of periodic titles are in accordance with List of Journals Indexed

in Index Medicus.

TABLES OF CONTENTS

Abbreviations and Acronyms List

Figures List

Tables List

Resumo

Abstract

1 INTRODUCTION ................................................................................................................ 1

2 OBJECTIVES..................................................................................................................... 11

2.1 Primary objective ................................................................................. 11

2.2 Secondary objectives ......................................................................... 11

3 METHOD ............................................................................................................................. 12

3.1 Study Design ....................................................................................... 12

3.2 Participants .......................................................................................... 12

3.3 Prophylaxis .......................................................................................... 13

3.4 Immunosuppression ............................................................................ 14

3.5 Microbiology ........................................................................................ 14

3.5.1 Surveillance Culture .................................................................. 14

3.5.2 Processing of surveillance culture samples .............................. 15

3.5.3 Culture for diagnosis of infection ............................................ 17

3.5.4 Molecular typing – Pulsed Field Electrophoresis ................... 18

3.6 Definitions ............................................................................................ 21

3.6.1 Multidrug-resistant bacteria definition ........................................ 21

3.6.2 Diagnostic Criteria for bacterial colonization .......................... 22

3.6.3 Criteria for diagnosis of healthcare-associated infection ....... 22

3.6.4 Definition of acquisition of Multidrug-resistant bacteria ......... 23

3.6.5 Criteria for diagnosis of acute cellular rejection ..................... 24

3.6.6 Criteria for severity of liver failure: .......................................... 24

3.7 Statistical treatment ............................................................................ 25

3.7.1 Performance of surveillance culture: ......................................... 25

3.7.2 Outcome variables ................................................................... 25

3.7.3 Variables analyzed .................................................................. 26

3.7.4 Statistical analysis ................................................................... 28

4 RESULTS............................................................................................................................ 30

4.1 Characterization of the population ....................................................... 30

4.2 Surveillance cultures ............................................................................ 34

4.3 Identification of KPC-producing Enterobacteriaceae ........................... 36

4.4 Analysis of risk factors for Multidrug-resistant Gram-negative bacteria acquisition after liver transplantation ...................................... 37

4.4.1 Analysis of risk factors for carbapenem-resistant K.

pneumoniae acquisition after liver transplantation .................... 39

4.4.2 Analysis of risk factors analysis of carbapenem-resistant A.

baumannii acquisition after liver transplantation ........................ 41

4.4.3 Analysis of risk factors analysis of carbapenem-resistant P.

aeruginosa acquisition after liver transplantation ...................... 44

4.5 Healthcare-associated infections (HAI) after liver transplantation ....... 46

4.5.1 Surgical site infections .............................................................. 48

4.5.2 Bloodstream Infections after Liver Transplantation ................... 50

4.5.3 Lower respiratory tract infection after liver transplantation ........ 51

4.5.4 Other types of infection after liver transplantation ..................... 52

4.6 Analysis of risk factors for healthcare-associated infection by MDR GNB after liver transplantation ............................................................. 53

4.6.1 Analysis of risk factors analysis of healthcare-associated

infection by carbapenem-resistant K. pneumoniae after liver

transplantation ........................................................................... 56

4.6.2 Analysis of risk factors for healthcare-associated infection by

carbapenem-resistant A. baumannii after liver transplantation . 58


carbapenem-resistant P. aeruginosa after liver

transplantation ........................................................................... 60

4.7 Evaluation of strains clonality through PFGE ...................................... 62

4.8 Survival analysis .................................................................................. 66

5 DISCUSSION..................................................................................................................... 69

6 BIBLIOGRAPHY ............................................................................................................... 84

7 APPENDIX

Abbreviations and Acronyms List

SOT – Solid organ transplantation

CMV – Cytomegalovirus

EBV – Epstein-Barr virus

HAI – Healthcare-associated infection

MDR – Multidrug-resistant bacteria

LT – Liver Transplantation

MRSA – Methicillin-resistant Staphylococcus aureus

MELD – Model for End-Stage Liver Disease

VRE – Vancomycin-resistant enterococci

GNB – Gram-negative bacilli

ESBL – Extended spectrum beta-lactamase

CRE – Carbapenem-resistant Enterobacteriaceae

CRPA – Carbapenem-resistant Pseudomonas aeruginosa

CRKP – Carbapenem-resistant Klebsiella pneumoniae

CVC – Central venous catheter

BHI – Brain Heart Infusion

CLSI – Clinical Laboratory Standards Institute

PFGE – Pulsed-field Gel Electrophoresis

SSI-OE – Organ-space Surgical Site Infection

SSI-I – Incisional surgical site infection

BSI – Bloodstream infection

UTI – Urinary tract infection

RR – Relative risk

CI – Confidence interval

OR – Odds Ratio

Figures List

Figure 1 – Distribution of PFGE pulsotype of carbapenem-resistant A.

baumannii strains from November 2009 to November 2011 ......... 62

Figure 2 – Dendogram of carbapenem-resistant A. baumannii strains from

eight patients who acquired CRAB before LT and developed

infection by CRAB after LT ............................................................ 63

Figure 3 – Distribution of PFGE cluster of carbapenem-resistant blaKPC-

positive K. pneumoniae strains from January 2010 to November

2011 .............................................................................................. 64

Figure 4 – Dendogram of carbapenem-resistant K. pneumoniae isolated

from Liver transplant patients from November 2009 to

November 2011 ............................................................................. 65

Figure 5 – 60-day survival curve adjusted according to LT surgery duration

and post-LT dialysis, categorized by type of microorganism

isolated from HAI after LT.............................................................. 68

Tables List

Table 1 – Baseline diseases of 195 patients submitted to liver

transplantation ............................................................................. 31

Table 2 – Demographic and clinical characteristics of 195 patients

submitted to liver transplantation ................................................. 32

Table 3 – Sites, microorganisms and sensitivity profile of 50 infections

identified in up to ten days before liver transplantation ................. 33

Table 4 – Positivity rate of surveillance culture by site of harvest from the

different Multidrug-resistant Gram-negative bacteria in 180

patients submitted to liver transplantation ................................... 35

Table 5 – Performance of surveillance culture of different MDR GNB in

180 patients submitted to liver transplantation ............................ 35

Table 6 – Distribution of blaKPC gene positivity rate among strains of

carbapenem-resistant Enterobacteriaceae isolated from

surveillance culture...................................................................... 36

Table 7 – Univariate analysis of risk factors for acquisition of MDR GNB

after LT in 105 patients – categorical variables ........................... 37

Table 8 – Univariate analysis for risk factors for acquisition of MDR GNB

after LT in 105 patients – continuous variables ........................... 38

Table 9 – Multivariate analysis of risk factors for acquisition of MDR

GNB after LT in 105 patients ....................................................... 38

Table 10 – Univariate analysis of risk factors for acquisition of

carbapenem-resistant K. pneumoniae after LT in 172 patients –

categorical variables.................................................................... 39


carbapenem-resistant K. pneumoniae after LT in 172 patients

– continuous variables ................................................................. 40

Table 12 – Multivariate analysis of risk factors for acquisition of

carbapenem-resistant K. pneumoniae after LT in 172 patients....... 40


carbapenem-resistant A. baumannii after LT in 171 patients –



carbapenem-resistant A. baumannii after LT in 171 patients –

continuous variables.................................................................... 43


carbapenem-resistant A. baumannii after LT in 171 patients ...... 43


carbapenem-resistant P. aeruginosa after LT in 190 patients –



carbapenem-resistant P. aeruginosa after LT in 190 patients –

continuous variables.................................................................... 45


carbapenem-resistant P. aeruginosa after LT in 190 patients ....... 45

Table 19 – Distribution of the sites of 201 healthcare-associated infections

after liver transplantation ............................................................. 46

Table 20 – Distribution of microorganism isolated from 201 healthcare-

associated infections after liver transplantation ........................... 47

Table 21 – Microorganisms isolated from post-transplant organ space

surgical site infections, percentage of bacteremia and

resistance profile. ........................................................................ 49

Table 22 – Microorganisms isolated from post-transplant bloodstream

infections and resistance profile .................................................. 50

Table 23 – Microorganisms isolated from lower respiratory tract infection,

secondary bacteremia and resistance profile. ............................. 51

Table 24 – Microorganisms isolated from post-transplant urinary tract

infections and resistance profile. ................................................. 52

Table 25 – Microorganisms isolated from soft tissue infections and

resistance profile ......................................................................... 53

Table 26 – Univariate analysis of risk factors for healthcare-associated

infection by MDR GNB after liver transplantation in 195

patients – categorical variables ................................................... 54


infection by MDR GNB after liver transplantation in 195

patients – continuous variables ................................................... 55

Table 28 – Multivariate analysis of risk factors for healthcare-associated

infection by MDR GNB after LT in 195 patients ........................... 55



transplantation in 194 patients – categorical variables ................ 56


infection by carbapenem-resistant K. pneumoniae after LT in

194 patients – continuous variables ............................................ 57



transplantation in 194 patients .................................................... 57


infection by carbapenem-resistant A. baumannii after liver




transplantation in 193 patients – continuous variables ................ 59



transplantation in 193 patients – categorical variables ................... 59


infection by carbapenem-resistant P. aeruginosa after liver




transplantation in 195 patients – continuous variables ................ 61




Table 38 – Univariate analysis of risk factors for death in the 60 first days

after liver transplantation, among 195 patients – categorical

variables ...................................................................................... 66

Table 39 – Univariate analysis of risk factors for death in the 60 first days

after liver transplantation, among 195 patients – continuous

variables ...................................................................................... 67

Table 40 – Multivariate analysis of risk factor for death in the 60 first days

after liver transplantation, among 195 patients ............................ 67

RESUMO

Freire MP. Aquisição de bactéria gram-negativa multidroga resistente antes do transplante de fígado: o impacto no desfecho [Tese]. São Paulo: Faculdade de Medicina, Universidade de São Paulo; 2017.

As infecções em pacientes submetidos a transplantes de órgãos sólidos são importante causa de morbidade, além de serem definidoras da sobrevida desta população. A maioria das infecções que ocorre nos dois primeiros meses pós-transplante é relacionada à assistência à saúde (IRAS). O objetivo deste trabalho é identificar fatores de risco para IRAS por bactérias Gram-negativas (BGN) multi-droga resistentes (MDR) em pacientes submetidos a transplante de fígado (TF), nos dois primeiros meses após o transplante. Os objetivos secundários são: identificar fatores de risco para aquisição por MDR GNB em pacientes submetidos a TF, e determinar o impacto das IRAS por MDR GNB na sobrevida desses pacientes. Foram avaliados os TF consecutivos realizados em pacientes adultos no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) no período de novembro de 2009 a novembro de 2011. A vigilância microbiológica foi realizada no dia do TF, e semanalmente até a alta hospitalar ou 60 dias após o transplante. Os sítios de coleta f o r a m swab de orofaringe ou secreção traqueal, swab retal e swab axilar. Foram pesquisadas as seguintes bactérias: A. baumannii. P. aeruginosa e Enterobactérias resistentes a carbapenêmico, e K. pneumoniae e E. coli produtoras de betalactamase de espectro estendido (ESBL). Posteriormente, as amostras clínicas foram comparadas com as cepas da mesma espécie isoladas em culturas de vigilância por tipagem molecular. A análise de fatores de risco foi realizada por tipo de infecção e espécie de bactéria. Na análise estatística utilizou-se o teste qui-quadrado ou teste exato de Fisher para variáveis dicotômicas, e teste de Mann-Whitney para variáveis ordenáveis. A análise multivariada foi realizada por regressão logística. A análise de sobrevida foi realizada por regressão de Cox. O nível de significância de P considerado foi 0,05. Foram realizados, no período, 229 transplantes em 202 pacientes, e analisados 214 transplantes em 195 pacientes. O motivo de indicação do transplante mais frequente foi cirrose pelo vírus C, 33%. Foram identificados no período do estudo 110 pacientes (56,4%) com IRAS pós-TF, e um total de 201 infecções. Em 76,3% dos pacientes com IRAS (84/110) foi isolado MDR GNB em alguma amostra clínica relacionada à infecção. Os dois principais sítios de infecção foram infecção de sitio cirúrgico (32%) e infecção primária de corrente sanguínea (27%). Os dois microrganismos mais frequentemente isolados das IRAS foram A. baumannii e K. pneumoniae, e a proporção de infecções por cepas resistentes a carbapênemico foi, respectivamente, 100% e 48,9%. Os fatores de risco para infecções por MDR GNB pós-TF foram: retransplante precoce, volume de concentrados de hemácias transfundidos no intra-operatório da cirurgia do TF, colonização por MDR GNB no pré-transplante, tempo prolongado de internação em UTI e tempo prolongado de

isquemia fria. Cento e cinco pacientes adquiriram algum MDR GNB nos 60 dias pós-TF, e o único fator de risco detectado para aquisição de MDR GMB no pós-TF foi tempo prolongado de sonda vesical de demora. A análise de clonalidade demonstrou que as cepas de MDR identificadas pré-TF eram fortemente relacionadas às cepas isoladas das infecções no pós-TF para A. baumannii e K. pneumoniae resistente a carbapenêmico. As infecções por MDR GNB apresentaram uma tendência a aumentar o risco de óbito nos 60 primeiros dias pós-TF, mas esta.

Descritores: transplante de fígado; bactéria multi-droga resistente; fator de risco, mortalidade; bacilo Gram-negativo; resistência a carbapenêmico

ABSTRACT

Freire MP. Multidrug-resistant Gram-negative bacteria acquired before liver transplantation: the impact on the outcome [Thesis]. São Paulo: "Faculdade de Medicina, Universidade de São Paulo"; 2017. Bacterial infections among patients submitted to liver transplantation (LT) are an important cause of morbidity and have huge impact on patients' survival. The majority of infections in the first two months after LT are related to healthcare assistance. The aim of this study has been to identify risk factors for healthcare-associated infections (HAI) caused by multidrug-resistant Gram-negative bacteria (MDR GNB) in liver transplant patients in the first two months after LT. The secondary aims have been to identify risk factors for acquisition of MDR BGN among liver transplant patients and analyze the survival rate during the first two months after LT. We analyzed consecutive liver transplantations performed at Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) from November 2009 to November 2011. Surveillance cultures were performed on a weekly basis, starting on the day of the LT until the hospital discharge or 60 days after the LT. We collected surveillance cultures through swab from oropharynx (or tracheal secretion), axillary and inguinal rectal sites. We surveyed the following bacteria: carbapenem-resistant A. baumanni, P. aeruginosa, Enterobacteriaceae, ESBL-producing K. pneumoniae, and E. coli. The strains isolated from surveillance culture were compared to strains isolated from clinical cultures through PFGE. The risk factor analysis was performed for each type of MDR bacterium for risk of colonization and infection. The statistical analysis was carried out for dichotomous variables using chi-square tests or Fisher's exact tests when appropriate; Mann-Whitney tests were used for continuous variable and step-wise logistic regression was used for multivariate analysis. The survival rate analysis was performed using Cox regression. The significant value of P was 0.05. During the study period, 229 liver transplantations were performed in 202 patients and we analyzed 214 LT performed in 195 patients. The main baseline disease that warranted LT was virus C cirrhosis, 33%. 110 (56.4%) patients developed healthcare-associated infections after the LT and a total of 201 infections were identified; 84 (76.3%) patients had MDR GNB isolated from clinical cultures related to HAI. Surgical wounds (31%) and primary bloodstream (27%) were the most prevalent infection sites. The risk factors for HAI by MDR GNB after the LT were: re-transplantation, volume of blood units transfused during the LT surgery, colonization by MDR GNB before the LT, prolonged time of ICU stay, and prolonged time of cold ischemia. 105 patients acquired MDR GNB during the first 60 days after the LT; the only risk factor identified was the prolonged use of urinary drain. The clonal analysis showed that strains isolated in the period before the LT were closely related to strains isolated from clinical culture after the LT for carbapenem-resistant A. baumannii e K. pneumoniae. The infections by MDR GNB have been shown to increase the risk of death in the first 60 days after LT. Descriptors: liver transplantation, multidrug-resistant bacteria, risk factor, mortality, Gram-negative bacillus, carbapenem resistance

1 INTRODUCTION

Infections in patients undergoing solid organ transplant (SOT) are an

important cause of morbidity and have a decisive role in survival of this

population. Those infections may be didactically divided into three periods,

in which the incidence of different pathogens varies according to the state of

immunosuppression and environmental exposures: (1)

1. First or early period - comprises the first thirty days after the

transplantation, when most infections occur. Risk factors are mainly

related to the surgical procedure and the postoperative hospital stay.

The most common agents are bacteria commonly related to

healthcare-associated infection (HAI) and fungi that cause HAI,

especially Candida species. Herpes simplex virus infections may

already occur at this stage.

2. Second period - extends from the second to the sixth month post-

transplantation, and the infections are in general related to the

immunosuppression state. In this period there can occur opportunistic

infections caused by cytomegalovirus (CMV), Pneumocystis jiroveci

pneumonia and infections by Aspergillus species. Besides that, latent

infections such as tuberculosis can be reactivated.

Introduction 2

3. Third or late post-transplantation period - after six months of

transplantation, the majority of the patients are prone to lower levels

of immunosuppression. At this stage, infections are usually the

same as those acquired in the community by the immunocompetent

population. There are, however, some exceptions, such as those

patients who develop late acute rejection or chronic rejection or

other situations requiring an increase in immunosuppression, which

increases the risk of opportunistic infections. Development of

chronic infections such as hepatitis B and C and lymphoproliferative

diseases associated with Epstein-Barr virus (EBV) can also occur.

Currently, some late opportunistic infections such as CMV infection

can occur in this period if prophylaxis has been given earlier.

The risk of opportunistic infections is much more dependent on the

area under the curve of immunosuppression over time than on the dose of

immunosuppressant drug. Thus, in the early period the majority of infections

are not caused by opportunistic pathogens but are mainly related to patient

care and the surgical procedure, i.e., HAI. (2) (1)

Most infections in patients undergoing SOT occurred in the early

period. In 95% of the cases, the causative agent is a bacterium, and it is

estimated that about 60% of the infections are caused by multidrug-resistant

bacteria (MDR). (1) (3)

In this first period, patients undergoing liver transplantation (LT) are

particularly susceptible to HAI compared to patients who have been

Introduction 3

submitted to other SOT. There are certainly many factors that can contribute

to this fact: the complexity of the procedure, prolonged hospital and

intensive care stay, immunosuppression caused by baseline diseases,

frequency of renal failure, malnutrition and hepatic failure by itself. It is

expected that up to 68% of the patients will develop some kind of infection

during the first months after LT compared with 47% of kidney transplant and

54% of lung transplant recipients. (1) (2) (4)

Among bacterial infections after LT, those caused by Gram-positive

bacteria have been described as the most frequent ones in many studies.

The most commonly isolated agent was S. aureus, with an average incidence

of 25% and a high proportion of infections caused by methicillin-resistant

strains(MRSA). (5) (6) The prevalence of MRSA varies from 1.3% to 22.7% in

the post-LT period. (7) Among the patients undergoing LT at the Hospital das

Clínicas of the University of São Paulo Medical School (HCFMUSP), MRSA

have also been identified as the most frequent pathogens in the period from

2000 to 2005, accounting for 16% of the infections in the first month post-

transplant, with 94% of methicillin resistance. (8) Risk factors for MRSA

infection after LT are well established, and among them, the most important

is the previous colonization, both pre- and post-LT. (9) (10) It is estimated that

pre-transplant colonization increases the risk of infection by MRSA in the

post-transplant period in more than five times. (7) Another risk factor identified

in some studies has been the degree of hepatic impairment, measured by

MELD (Model for End-Stage Liver Disease) or prothrombin time test. (10)

Screening all patients, decolonization of carriers, and maintenance of straight

Introduction 4

contact precautions measures for hospitalized, colonized patients have been

shown to reduce the incidence of MRSA infections. Singh et al have shown a

drop from 40% to 4% in the rate of MRSA infection with the implementation of

this bundle of measures. (11)

Enterococcus spp. is a gram-positive bacterium with an increasing

prevalence among patients submitted to LT, the incidence ranging from 1 to

16%, and vancomycin resistance in up to 40% depending on the

epidemiological resistance profile of the transplant center. The prevalence

of vancomycin-resistant Enterococcus (VRE) post-transplantation is on

average 11.9%, ranging from 6.8% to 18.2%. (7) In our service this agent

was identified in 11% of cases of the infections in the first month after LT,

and vancomycin resistance was detected in 42% of the cases. (12) The

predisposing factors to Enterococcus spp. infections are the prior use of

antibiotics, biliary-digestive anastomosis and surgical complications related

to the biliary tree. (12) (13) (14). Colonization by VRE significantly increases the

risk of infection by this pathogen, and it is estimated that the probability of

VRE-colonized LT patients developing a VRE infection in the post-LT period

is five times higher than that of non-colonized patients. (14)

The prevalence of infections caused by Gram-negative bacilli (GNB)

has been increasing in recent years. Singh et al have identified an increase in

Gram-negative bacteremia cases, justified in part by the better management

of fungal and viral infections surveillance. At our center, Gram-negative

infections accounted for 47% of HAI in the first month post-LT. (15) (8) (16) Other

studies have also pointed to GNB as the predominant causative pathogen

Introduction 5

among post-LT bacteremic infections. Currently, Gram-negative infections

may account for up to 65% of post-LT bacterial infections. (17)

In addition to the increase in the prevalence, certain species with

reduced susceptibility to many classes of antimicrobial agents are on the

increase in this group of bacteria. (18) (19) (20) Singh et al have found that 54% of

the Gram-negative isolates in post-LT infections are classified as MDR, and

that there has been an increase in extended-spectrum beta-lactamase-

producing (ESBL) strains over time. The one-year mortality rate was

significantly higher for patients with infections caused by MDR bacteria. (21)

High morbidity and mortality rates of Gram-negative HAI can be

associated with this frequent multidrug resistance profile, which increases

the probability of errors in empiric treatment, and reduces the range of

options available for effective antimicrobial therapy. (6)

Pseudomonas aeruginosa is often isolated after LT and is a frequent

etiologic agent of pneumonia. (6) (17) Some reports claim that patients submitted

to LT are three times more susceptible to developing P. aeruginosa

bacteremia than other SOT patients. (22) Among patients submitted to LT at

HCFMUSP, P. aeruginosa was the fourth most prevalent HAI-causing

Gram-negative bacillus, but remained the most common etiologic agent of

pneumonia. (8)

Although P. aeruginosa is frequently associated with pneumonia,

some studies have reported that this bacterium is also commonly isolated

from intra-abdominal infections, and biliary intercurrences after LT are risk

Introduction 6

factors for infection by that etiologic agent. (23) (24) Other risk factors

described are: the intensive care unit (ICU) stay, re-transplantation and

bloodstream infection (BSI) associated with health assistance. (25)

Solid Organ Transplantations were previously described as risk factors

for infection by carbapenem-resistant P. aeruginosa (CRPA). (26) Other risk

factors often identified for HAI by CRPA in the general population are:

prolonged hospital stay and antibiotic use, common situations found in Liver

transplant patients. (27) (28) A Brazilian study has identified this microorganism

as the most frequent MDR bacteria in LT. (29) The resistance of P. aeruginosa

to carbapenem in LT recipients is described in the literature as ranging from

30 to 83%. (21) (30) A cohort study of 49 cases of P. aeruginosa bacteremia in

SOT patients identified the following factors associated with CRPA:

re-transplantation, septic shock, nosocomial acquisition. (31)

Infections by Acinetobacter baumannii in LT recipients range widely in

their incidence depending on the institution. Torre-Cisneros et al identified A.

baumannii in 7.6% of BSI. (5) Among the patients submitted to LT at

HCFMUSP, A. baumannii was the second most common Gram-negative

agent causative of HAI in the first post-LT period, corresponding to 12% of

the isolated microorganisms. (8) In that population, the A. baumannii was the

most frequent pathogen isolated from surgical site infections, and 94% of

the strains were resistant to carbapenem. (32)

The lung is the most common site of infection by this agent in SOT

patients. However, particularly in LT patients, intra-abdominal infections are

the predominant site in most series. (33) (34) (35)

Introduction 7

Previously documented risk factors for HAI by A. baumannii in

the general population are: disease severity, surgical procedures,

immunosuppressive drug use, previous antibiotic use and ICU stay. (36)

Those characteristics are usually identified in patients undergoing LT, and

so we expect that in services with a high prevalence of A. baumannii

infections, the LT population is an important group that develops HAI

caused by this agent. Specific risk factors for A. baumannii infection in

patients submitted to SOT described in the literature are: high pre-LT MELD

score, prior use of carbapenem, use of central venous catheters and

dialysis. Those studies, however, were not subjected to a multivariate

analysis to identify risk factors due to the small number of cases. (33) (37)

Klebsiella pneumoniae is the second most frequently identified Gram-

negative after LT in the literature, and the most frequent microorganism

identified at HCFMUSP. (8) Wade and colleagues, in a study of 284 liver

transplant patients identified infections by these microorganisms as a risk

for increased length of hospital stay. (38) Liver transplant recipients are

particularly susceptible to infections by Enterobacteriaceae. (30) In an

outbreak of ESBL-producing Escherichia coli (E. coli), 67% of the patients

were from an LT ward. (39) The intestine is the most frequent site of

colonization by Enterobacteriaceae and Liver transplant patients have an

increased risk of bacterial translocation due to frequent complications of

biliary tract and Roux-en-Y hepatojejunostomy anastomosis. Besides that,

LT patients are often exposed to antimicrobials, which increase the risk of

infections caused by ESBL-producing strains.

Introduction 8

Zhou et al identified 55 Enterobacteriaceae infections after LT. 32.4%

of them were ESBL-producing Enterobacter cloacae strains and 24% of

them were ESBL-producing AmpC. (40)

Infections caused by ESBL-producing Enterobacteriaceae are highly

prevalent among SOT recipients, approximately 10 to 50%. The mean

incidence is highly variable, and unlike other MDR bacterial infections,

infections by ESBL-producing Enterobacteriaceae are also described in

later periods after transplantation. (6) (41) (42) The single factor identified as a

risk of infection by ESBL-producing Enterobacteriaceae in transplant

patients is the need for post-transplant hemodialysis. (6)

Currently, Enterobacteriaceae resistance to carbapenem (CRE) is

being increasingly reported with a high worldwide prevalence of these

agents. (43) (44) This resistance can occur due to several mechanisms: AmpC

hyper-expression or class A ESBL (either TEM or SHV) associated with

changes in the membrane (permeability of the outer membrane or hyper-

regulation of efflux bombs), or the production of enzymes that hydrolyze

carbapenem (carbapenemases). The carbapenemases identified in

Enterobacteriaceae can be categorized as metallo-β-lactamases, extended-

spectrum β-lactamase oxicilinases or carbapenemases inhibited by

clavulanic acid. (19) Carbapenemases inhibited by clavulanic acid belong to

Ambler’s Class A, among which KPC are the most frequent enzymes. (19)

The mortality rate associated with CRE infections ranges between 28%

and 68%. The introduction of effective therapy usually suffers greater delay,

with a consequent more prolonged hospital stay after diagnosis. (44) (45) (46)

Introduction 9

For KPC-producing K. pneumoniae (KPKPC), there are reports of an

increased risk of death by up to four times, and the risk factors associated

with higher mortality rates are high Acute Physiology and Chronic Health

Evaluation II (APACHE II) score, inadequate initial antibiotic therapy, age,

and septic shock at diagnosis of infection. (44) (47) (48) (46) (49) (50) (51)

The risk factors identified for CRE infections are an increased number

of invasive procedures, immunosuppression, use of cephalosporins,

carbapenems, anti-pseudomonas penicillin and fluoroquinolone, high APACHE

II score, mechanical ventilation, length of hospital stay, patient’s severity rating,

ICU stay, transplant of hematopoietic stem cells and SOT. (48) (47) (52) (53) (54)

Although patients submitted to SOT are at a higher risk for CRE

infections, there is scarce data in the literature on this specific population.

A KPKPC outbreak report in a hospital in São Paulo city described the

evolution of 10 SOT recipients: four kidney transplants, four LT and two

heart transplants, with an associated mortality rate of 50%. (55)

The incidence of KPKPC in patients undergoing LT varies from zero

to 25% of the bacterial infections in the first year post transplant.

Bloodstream infections and intra-abdominal infections are the two most

common types of infection. These infections have a significant impact on

the patient’s survival, and an associated mortality rate that ranges from 42%

to 71%. (55) (56) (57) (58)

In the Liver Transplant Service of the HCFMUSP, the carbapenem-

resistant K. pneumoniae infections were more frequent in intra-abdominal

Introduction 10

sites, mainly surgical site infections (SSI) with a high incidence of

bacteremia, in 46% of the cases. Mortality rate was 27%, and the risk

factors identified for carbapenem-resistant K. pneumoniae (CRKP)

infection were previous colonization and the need for dialysis. In the

literature the risk factors identified for infection by CRKP in LT recipients

are: mechanical ventilation for more than 48 hours, recurrent hepatitis C

virus infection, dialysis, high MELD score, Roux-en-Y biliary anastomosis,

biliary fistula, and colonization by CRKP. (59) (60)

Apparently, infections caused by MDR-GNB in patients undergoing

LT are strongly influenced by the frequency and sensitivity profile of the

microorganisms found not only in the transplant facilities, but also in all the

other hospital areas. Therefore, considering the growing incidence of MDR-

GNB, it seems essential that the analysis of risk factors for MDR-GNB

infections include active screening of colonization. The role and impact of

colonization as a risk for infection by these agents are not yet definitely

established in the literature. A better understanding of the epidemiology of

infections by MDR-GNB can contribute to the development of more effective

control strategies, as well as the most appropriate management of patients

with suspected infection by these agents.

11

2 OBJECTIVES

2.1 Primary objective

The primary objective was to analyze the impact of MDR-GNB

acquisition prior to LT on the incidence of healthcare-associated infections

by MDR-GNB in the first two months after LT.

2.2 Secondary objectives

To identify risk factors for healthcare-associated infection by

MDR-GNB in the first two months after LT.

To identify risk factors for MDR-GNB acquisition in the first two

months after LT.

To identify risk factors for acquisition and infection by the following

species of MDR-GNB (carbapenem-resistant P. aeruginosa, A.

baumannii, and Enterobacteriaceae) in the first two months after LT.

To analyze the impact of MDR-GNB infection in the 60-day

mortality rate after LT.

12

3 METHOD

3.1 Study Design

This was a prospective cohort study without intervention.

3.2 Participants

We evaluated consecutive liver transplants performed in LT patients

at HCFMUSP from October 2009 to October 2011. The exclusion criterion

was death in up to 48 hours after LT.

The liver transplantation service of HCFMUSP has been in activity

since 1988. During the study period an average of 100 transplants a year

were performed.

Data collection was prospective, using the following sources: the

patient's medical record, the records of the LT infectious diseases team,

infection control service database and the record of post-LT follow-up.

If the patient were discharged in less than two months after the liver

transplantation, the monitoring was conducted in an LT outpatient care setting.

Method 13

The HAI data and incidence of MDR bacteria were monitored monthly

by the hospital’s infection control service and surveillance cultures for CRE,

VRE and carbapenem-resistant Acinetobacter baumannii (CRAB) were

collected at admission and on a weekly basis for all patients in the liver

transplant ward and ICU.

3.3 Prophylaxis

The surgical prophylaxis used during the study period consisted of

a 48-hour administration of ampicillin plus cefotaxime. In the case of

patients undergoing LT during the treatment of infections, we used the

therapeutic antibiotic for prophylaxis provided its spectrum was similar to or

wider than that of the scheme used for surgical prophylaxis. For

prophylaxis in LT recipients whose donors were under infection treatment,

we used the same antimicrobial as the one received by the donor, or a

similar one, provided its spectrum was similar to or wider than that of the

scheme used for surgical prophylaxis.

Antifungal prophylaxis was performed for all patients with fulminant

hepatitis, dialysis indication, re-transplantation or treatment for acute cellular

rejection. Patients with indication for antifungal prophylaxis received

amphotericin B for 7 days.

Method 14

3.4 Immunosuppression

The standard immunosuppression regimen was performed with

tacrolimus associated with corticosteroids. The initial dose of tacrolimus was

0.3 mg/kg/day given every 12 hours (approximately 1 mg PO bid) for the first

days post-transplant and was adjusted to achieve a blood concentration of 8-

12 in the first three months. The induction immunosuppression was performed

with 1g of methylprednisolone administrated during the anhepatic phase.

3.5 Microbiology

3.5.1 Surveillance Culture

Cultures for identification of colonization by CRE, ESBL-producing

Enterobacteriaceae, CRPA and CRAB were performed immediately before

transplantation and weekly after LT while the patient was hospitalized. If the

patient happened to be re-hospitalized within two months after LT, the collection

of cultures was again performed at readmission and on a weekly base.

The materials for surveillance cultures were collected by oropharyngeal

swab or tracheal aspirates (if the patient was on mechanical ventilation),

and by axillary and inguinal-rectal swab.

Method 15

The transport of the material was carried out in dry sterile bottles in

the case of tracheal aspirates, or culture transport swab (Mueller Hinton) for

oropharyngeal, axillary and inguinal-rectal samples.

The first surveillance culture (SC) was collected on the transplantation

day. SC samples were collected twice a week, on Mondays and Thursdays,

so that the second week SC was collected in an interval that varied from six

to nine days from the first collection. The main researcher performed all the

SC collections.

3.5.2 Processing of surveillance culture samples

The SC were processed at HCFMUSP’s bacteriology and clinical

research laboratory (LIM 54).

Surveillance cultures were plated on selective media containing either

imipenem (1ml of Brain Heart Infusion broth (BHI) with a 10-µg-imipenem

disk) or ceftriaxone (1,8ml of BHI with a 30-µg-Ceftriaxone disk).

For identification, the isolated bacterial colonies were subjected to

optical microscopy and biochemical tests. The identification of the colonies

was carried out after the analysis of bacterial growth in different culture

media, observing its characteristics of size, edge, elevation, color, density,

consistency and hemolysis on blood agar media.

Method 16

The Enterobacteriaceae were identified by IAL medium (modified

Rugai) and oxidase and citrate utilization tests. (61) Nonfermenting Gram-

negative bacilli were identified through biochemical tests.

Several colonies were used in identification and subsequently

subjected to sensitivity tests for confirmation of their phenotype.

Suspected colonies were characterized by a commercial microorganism

identification kit (API 20E; BioMérieux).

Disk-diffusion test (Kirby-Bauer method)

a) Preparation of the bacterial suspension and its inoculation on plates

A bacterial suspension at a scale of 0.5 McFarland (~ 1.5 x 108 CFU /

ml) was prepared by spectrophotometry, with the optical density of 0.08-0.1

at 625nm. A cotton swab was used to inoculate a homogeneous bacterial

suspension on a Petri dish containing Mueller-Hinton agar (diameter

150mm). The procedures followed in this step were performed according to

standardized rules for the document M2-A7. (62)

b) Application of the disks and incubation of the plates

After a period of 15 minutes, the antibiotic disks were applied to the

plates. The samples were incubated at 35°C for 16 to 20 hours in the

ambient atmosphere. The antibiotic discs were chosen according to the

bacteria identified, following the recommendations of the Clinical Laboratory

Standards Institute (CLSI). (63)

Method 17

c) Reading of the plates

The diameters (mm) of the inhibition halos were measured with the

aid of a ruler and compared to the cutoff points proposed by CLSI. (63) The

isolates were then characterized as either sensitive or resistant.

Confirmation of carbapenem sensitivity for P. aeruginosa and A.

baumannii was checked using the disk-diffusion method (Kirby-Bauer method)

and (mm) the inhibition zone diameters were measured with the aid of a ruler

and compared to the cutoffs standardized by the CLSI (64). We used the E-test

for confirmation of strains with MIC ≥2 mg / ml for carbapenem.

The detection of ESBL was carried out using the disk-diffusion

method, Ceftazidime and Cefotaxime for screening, followed by a

combination of Disc Test (with or without the addition of clavulanic acid),

and E-test strips when necessary (Ceftazidime and Cefotaxime with or

without clavulanic acid).

3.5.3 Culture for diagnosis of infection

Cultures for diagnosis of infection were performed as part of the health

assistance routine and collected according to indication after evaluation by

the LT infectious diseases team.

The identification and sensitivity tests of the isolated microorganisms

were performed using the CLSI methodology described above. (64)

Method 18

The identification and initial susceptibility testing of microorganisms were

performed by automated method (Vitek - BioMérieux Marcy l'Etoile, France).

The cultures of clinical samples were initially processed at

HCFMUSP’s Microbiology Laboratory of the Central Laboratory Division,

and then forwarded to the LIM 54 for further investigation.

Cultures of interest for this project that were sent to the LIM 54 were

isolated from ESBL-producing Klebsiella pneumoniae, ESBL-producing E.

coli, cephalosporin-resistant Enterobacter spp., CRPA, CRAB and CRE.

The sources of interest were surgical sites (wound secretion or ascites fluid),

pneumonia (tracheal aspirate or bronchoalveolar lavage) and bloodstream

(blood culture).

All carbapenem-resistant Enterobacteriaceae strains isolated via

surveillance or clinical culture were submitted to polymerase chain reaction

(PCR) for the blaKPC.

3.5.4 Molecular typing – Pulsed Field Electrophoresis

The tests for molecular typing were performed at the LIM 54.

Microorganims phenotypically identified as CRKP, CRAB and CRPA were

analyzed by pulsed-field gel electrophoresis (PFGE).

Method 19

3.5.4.1 Protocol for molecular characterization by pulsed-field

gel electrophoresis (PFGE)

Molecular analysis was performed by electrophoresis of pulsed-field

gels, according to the protocols described by Ridley and Kaufmann. (65) (66)

Preparation of the bacterial suspension

Bacteria were subcultured on 5% sheep-blood agar medium and

incubated for 18-24 hours at 35 ° C 2°C. Three to five colonies were

transferred into tubes containing 3 ml of broth and incubated at 35 ° C 2 ° C,

overnight. They were transferred into a previously weighed microtube, and

extracted about 2 ml of the broth bacterial growth. The micro centrifuge tubes

were centrifuged for 20 minutes at 11,000 rpm. The supernatant was discarded

and the sediment was washed three times using 1 ml of sterile saline solution.

After the last wash, the supernatant was discarded and the microtube was

weighed on an analytical balance. The bacterial mass was calculated and then

an appropriate volume of 25 mm EDTA pH 8.0 solution was added to give the

bacterial suspension a resulting concentration of 100g/L.

Preparation of agarose blocks

DNA blocks were obtained by mixing 225 L of TEN buffer solution

(100 mM of Tris, pH 7.5; 100 mM of EDTA; 150 mM of NaCl) plus 25 L of

the bacterial suspension with 250 L of agarose 2% in 0.5X TBE buffer

(0.089 M of Tris, 0.089 M of boric acid, 0.002 M of EDTA). This mixture was

poured into specific molds and chilled at 4 ºC for 30 minutes.

Method 20

Bacterial DNA extraction step

The agarose blocks were removed from the molds and incubated in

EC 2ml of buffer (6 mM of Tris, pH7.5; 1 M of NaCl; 0.01 M of EDTA, 0.5%

Sarkosyl, 0.2% deoxycholate) for 5 hours at 37°C under gentle agitation.

After that time period, the buffer was removed and CHEF TE-buffer (2 mL)

(0.1 M of Tris, pH 7.5; 0.1 M of EDTA) was added. The latter buffer was

washed and then the blocks were treated with a solution with a final

concentration of proteinase K (Invitrogen Life Technologies, Carlsbad, USA)

at 1.0 mg / mL in ES buffer ( 0.4M of EDTA, pH9,3, 1.0% Sarkosyl),

overnight at 50°C. After this incubation period, the blocks were then washed

five times with 2 mL of CHEF-TE buffer at intervals of one hour under gentle

agitation. After the last wash, the blocks were stored at 4ºC.

Restriction enzyme step

Before performing the enzymatic treatment, agarose blocks were

washed five times with DNS buffer (0.1 M of Tris pH 8.0, 5 mM magnesium

chloride) with one-hour intervals between washes. For each species of

bacteria isolated a specific restriction enzyme was used.

Gel preparation

The treated wafers were dipped in agarose gel at 1% (TBE 0.5X).

Electrophoresis was performed using CHEF-DRII system (Bio-Rad,

Richmond, USA).

The patterns of electric current variation (time switch) were

established according to the microorganism studied. In the case of K.

Method 21

pneumoniae, race conditions were 5 to 30 seconds (initial and final switch

time), 6 V / cm (electric current) and 23 hours of run time. After the

electrophoresis, the gel was stained with ethidium bromide solution (1µg/ml)

for 40 minutes, bleached in distilled water for 40 minutes and photographed.

Image analysis

The PFGE images were processed and analyzed by Bioumerics

software version 7.1 (Applied Maths, Sint-Martens-Latem, Belgium). The

images were normalized using standard molecular markers and then the

band patterns were compared. The genetic similarity analysis was

performed using the Dice coefficient with a band position tolerance of 1.25%

and 0.5% optimization. Isolates were separated by similarity and grouped by

pair by the arithmetic mean method. Only the biggest bands of 45.5 kb were

included in the analysis.

3.6 Definitions

3.6.1 Multidrug-resistant bacteria definition

Multidrug-resistant bacterium was defined considering the following

resistance profiles:

• Carbapenem-resistant P. aeruginosa

• Carbapenem-resistant A. baumannii

Method 22

• ESBL-producing E. coli and K. pneumoniae

• Carbapenem-resistant Enterobacteriaceae

• Methicillin-resistant S. aureus

• Vancomycin-resistant Enterococcus

3.6.2 Diagnostic criteria for bacterial colonization

Colonization was defined as proliferation of microorganisms detected

on the patient’s surface or internally, without necessarily resulting in

detectable immune response, cellular damage or clinical symptoms.

The criteria used to define the patient only colonized by a MDR GNB

was the isolation of the agent in one or more clinical specimen samples or

surveillance cultures, and the exclusion of any active infection by this agent

at the time of collection.

3.6.3 Criteria for diagnosis of healthcare-associated infection

The diagnoses of bacterial and fungal infectious diseases were

performed through medical history, physical examination findings and

laboratory data, based on the criteria defined by the Centers for Disease

Control and Prevention (CDC / NHSN). (67)

Method 23

Post-LT healthcare-associated infections were those that occurred at

least 24 hours after the LT procedure. The cases of superficial fungal

infections and esophageal candidiasis were excluded.

The surgical site infections were categorized as superficial incisional,

when infection affected the skin and subcutaneous tissue; deep incisional,

when infection affected fascial or muscle layers; and organ space infection

when the infection involved any part of the anatomy other than the incision

that was opened or manipulated during the surgical procedure. (67)

The primary BSI was positive if patients had a recognized pathogen

isolated in one or more blood samples and this microorganism was not

associated with infection at another site. The BSI were defined as

associated with central venous catheter (CVC) if the patient had been in use

of the CVC for more than 2 days before the diagnosis of infection,

considering the 1st day the day of the implant. Infections with a single agent

in blood culture but with criteria for infection of other sites were defined as

secondary BSI. (67)

3.6.4 Definition of acquisition of multidrug-resistant bacteria

The acquisition of MDR GNB was characterized in the case of

patients who had not had either colonization or infection caused by a certain

agent in over two years, and the MDR GNB was isolated through clinical

culture or SC. during the two-month follow-up period after LT.

Method 24

3.6.5 Criteria for diagnosis of acute cellular rejection

Acute cellular rejection was defined as graft inflammation caused by

genetic mismatch between donor and recipient, affecting primarily

interlobular bile duct and vascular endothelium, including portal vein,

hepatic venules and occasionally hepatic artery and its branches. (68)

The diagnosis of rejection was histologically determined. The criterion

used was based on biopsy findings of at least two of the following:

predominantly mononuclear portal infiltrate with lymphocytes in blast phase,

neutrophils and frequent eosinophils and inflammation/injury of the bile

ducts; subendothelial inflammation of the portal vein or hepatic venule

terminals. (68) The classification used the Banff criteria. (68)

3.6.6 Criteria for severity of liver failure

MELD was used as a prognostic index for the assessment of liver

failure. The MELD was calculated immediately before the LT. The MELD is

a score that was initially used for patients with cirrhosis undergoing

transjugular intrahepatic portosystemic shunt, and then validated to assess

survival in patients with end-stage liver disease. The formula for the

calculation uses the total bilirubin levels, serum creatinine and International

Normalized Ratio (INR). (69)

Method 25

3.7 Statistical treatment

3.7.1 Performance of surveillance culture

For each SC collection site, the positivity rate was calculated as

follows: total number of positive SC tests for a given MDR GNB at a specific

collection site divided by the total number of positive SC tests for that MDR

GNB. For all patients, cultures were reviewed from three months before LT

to three months after LT.

The sensitivity rates was calculated following the formula: number of

patients with a positive SC test for a specific MDR GNB divided by the total

number of patients with a specific MDR GNB isolated in a clinical or

Surveillance Culture test.

3.7.2 Outcome variables

The outcome variables analyzed were:

• HAI by MDR in the first 60 days after LT;

• HAI by CRPA in the first 60 days after LT;

• HAI by CRAB in the first 60 days after LT;

• HAI by CRKP in the first 60 days after LT;

• Acquisition of MDR GNB in the first 60 days after LT;

Method 26

• Acquisition of CRPA in the first 60 days after LT;

• Acquisition of CRAB in the first 60 days after LT;

• Acquisition of CRKP in the first 60 days after LT;

• Death in the first 60 days after LT.

3.7.3 Variables analyzed

The receptor-related independent variables were:

• Continuous: Length of pre-transplant hospital stay in days, age in

years, pre-transplant functional MELD score, duration of central

venous catheter use in days, duration of urinary catheter use in

days, duration of mechanical ventilation use in days, duration of

intra-abdominal drain use in days, days of parenteral nutrition, use

of antimicrobial in the three months from pre-LT to the outcome or

60 days after LT for non-cases.

• Categorical: acute cellular rejection, use of Pulse corticosteroid

therapy for treatment of rejection, infection within 10 days from

previous LT, post-transplant bacterial infection, acquisition of MDR

GNB after the transplant, acquisition of MDR GNB before LT, post-

transplant fungal infection, use of therapeutic antibiotics in the

three months prior to the LT, performing surgical procedures after

LT, re-transplantation less than two months from the first transplant,

performing of hemodialysis, type of immunosuppression used after

Method 27

transplantation - defined as standard scheme, the use of two

immunosuppressant drugs (tacrolimus and corticosteroids),

fulminant hepatitis as indication of LT, the occurrence of CMV

infection, biliary complications, and American Society

Anesthesiology score (ASA).

• The variables related to transplant were:

• Continuous: duration of transplant surgery in minutes, cold and

total ischemia time in minutes, numbers of blood cell units

transfused during the LT.

• Categorical: Senior Surgeon (main surgeon who performed the

transplant with over eight years’ experience), type of antibiotic

prophylaxis used in LT (standard prophylaxis was defined as

ampicillin and cefotaxime for elective surgery and clindamycin,

cefotaxime and amphotericin B for fulminant hepatitis), type of

biliary anastomosis (defined as standard choledocho-

choledochostomy).

Patients who had MDR GNB identified before LT were excluded from

the analysis of risk of acquiring MDR GNB. Patients who underwent LT

while under treatment for MDR GNB infection were excluded from the

analysis of risk factor for post-LT MDR GNB infection.

Method 28

Variables related to exposure time

For patients with positive outcome, we considered the exposure time

up to the outcome. For patients with a negative outcome, we considered the

total exposure time during the first 60 days after LT. For each outcome the

exposure time was calculated separately.

The variables were ordered after the initial analysis, and all of them

were converted into categorical. The cutoff point adopted was the median of

the variable of the cases considered, except in cases of surgical time and cold

ischemia time, where the cutoff was the 75th percentile of the total samples.

For the multivariate analysis, the choice between the continuous and

categorical variable of the same information was that with the less value of P.

3.7.4 Statistical analysis

Univariate analysis was performed using the chi-square test or

Fisher's exact test for categorical variables when indicated, and the Mann-

Whitney test for continuous variables, considering a significance level of P

= .05. For the multivariate analysis we used stepwise logistic regression.

Variables that reached a significance level of less than or equal to 0.2 in the

univariate analysis were included in the model, following the ascending

order of the P value. Variables were sustained in the model if they reached

a P <0.05 or reduced the log-likelihood ratio by -2. The mortality rate analysis

and survival plots were performed using Cox regression. The following

Method 29

variables were treated as time-depended variables: age, post-LT dialysis,

acute cellular rejection, HAI, HAI by MDR and CMV infection.

The inclusion criterion in the mortality rate multivariate analysis was P

value lower or equal to 0.2 in the univariate analysis. The inclusion of

mortality in the multivariate analysis model was also by Stepwise. The

statistical software used for performing the analysis was SPSS 17.0.

30

4 RESULTS

4.1 Characterization of the population

During the study period, 229 transplants were performed in 202

patients. Seven patients were excluded due to death in less than 48 hours.

A total of 214 LT in 195 patients were evaluated.

The most common baseline disease that motivated LT was cirrhosis

due to hepatitis C virus, in 33% of the cases. (Table 1)

Demographic and clinical characteristics of the 195 patients

evaluated are shown in Table 2.

Results 31

Table 1 – Baseline diseases of 195 patients submitted to liver transplantation

Baseline diseases N Proportion

Cirrhosis due to hepatitis C virus 51 26%

Alcoholic cirrhosis 26 13%

Cryptogenic cirrhosis 21 11%

Fulminant hepatitis 16 8%

Alcoholic and hepatitis C virus cirrhosis 13 7%

Cirrhosis due to hepatitis B virus 13 6%

Autoimmune hepatitis 6 3%

Primary biliary cirrhosis 5 3%

Hemochromatosis 5 3%

Budd-Chiari syndrome 4 2%

Non-alcoholic steatohepatitis 4 2%

Familial paramyloidosis 4 2%

Primary sclerosing cholangitis 3 2%

Ductopenic diseases 3 2%

Wilson diseases 3 2%

Biliary atresia 2 1%

Cholestatic syndrome 2 1%

Alfa-1-antitrypsin deficiency 2 1%

Schistosomiasis 2 1%

Intra-hepatic lithiasis 1 1%

Caroli’s disease 1 1%

Graft dysfunction 1 1%

Glycogenosis type ii 1 1%

Cirrhosis due to C and B virus 1 1%

Hemangioendothelioma 1 1%

Thrombosis of hepatic artery 1 1%

Alcoholic and hepatitis B virus cirrhosis 1 1%

Alagille syndrome 1 1%

Secondary biliary cirrhosis 1 1%

Results 32

Table 2 – Demographic and clinical characteristics of 195 patients submitted to liver

transplantation

Characteristic Proportion (N) Median (min-max)

Gender (Female) 35% (69) -

Live donor 12% (25) -

Age (years) - 49 (16-73)

Length of hospital stay (days) - 32 (3-146)

Hepatocellular carcinoma 31% (61) -

Early re-transplantation 13% (26) -

Liver-kidney transplant 6% (12) -

Reoperation after LT 32% (62) -

Length of hospital stay before LT (days) - 6 (0-67)

ASA score >3 54% (97) -

Surgery duration (minutes) - 488 (210-1330)

Choledocho-choledochostomy 94%(184) -

MELD score - 23 (6-53)

Total ischemia time (minutes) - 428 (65-960)

Cold ischemia time (minutes) - 397 (20-910)

Nº of blood units transfused in LT procedure - 2 (0-15)

Serum creatinine on the day of LT (mg/dl) - 1,1 (0.51-6.79)

Post-LT dialysis 58% (110) -

Immunosuppression therapy with 3 drugs 45% (87) -

Cytomegalovirus infection 23% (44) -

Bacterial infection 10 days before LT 25% (49) -

Acute rejection treatment * 24% (43) -

Length of stay in ICU (days) - 16 (1-74)

Colonization or infection by Vancomycin-resistant Enterococcus spp. 47% (92)

*Standard treatment rejection was done with pulsed methylprednisolone therapy

LT – liver transplantation; ASA – American society of anesthesiology; MELD – model for end-stage liver disease, ICU-

intensive care unit.

Forty-nine (25%) patients were diagnosed with bacterial infection 10

days before LT. Table 3 describes the agents and sites of infection of those

infections. One patient had two different infections in less than 10 days

before transplantation. The average number of days between infection and

Results 33

the transplant was four days. Thirty patients (15.4%) had a diagnosis of

infection within 48 hours before the transplantation.

Table 3 – Sites, microorganisms and sensitivity profile of 50 infections identified in up

to ten days before liver transplantation

Site of infection N (%) Nº Infections

with causative

agent identified

Microorganism Nº

N MDR

(%)

Bacteremia

Intra-abdominal infection 21 (43%) 18 (86%) K. pneumoniae 6 3 (50%)* 3 (50%)

Enterococcus spp. 7 4 (57%) 3 (43%)

E. cloacae 4 0 2 (50%)

E. coli 3 1 (33%)** 2 (67%)

S. aureus 3 3 (100%) 2 (67%)

C. tropicalis 3 0 2 (67%)

Streptococcus spp. 2 0 1 (50%)

P. aeruginosa 2 0 1 (50%)

Gram-negative bacillus

1 0 0

P. mirabilis 1 0 0

A. baumannii 1 1 (100%) 1 (100%)

C. freundii 1 0% 0

Bloodstream Infection 20 (41%) 20 (100%) S .aureus 4 3 (75%) 4 (100%)

Enterococcus spp. 3 0% 3 (100%)

A. baumannii 3 3 (100%) 3 (100%)

K. pneumoniae 3 2 (67%)*** 3 (100%)

E. coli 2 0 2 (100%)

SCN 2 0 2 (100%)

C. albicans 2 0 2 (100%)

E. cloacae 2 0 2 (100%)

C. glabrata 1 0 1 (100%)

E. aerogenes 1 0 1 (100%)

Pneumonia/Bronchitis 4 (8%) 4 (100%) K. pneumoniae 1 1 (100%)*** 0

A. baumannii 1 1 (100%) 1 (100%)

E. aerogenes 1 1 (100%) 1 (100%)

Urinary tract infection 4 (8%) 4 (100%) P. mirabilis 1 0% 0%

S. aureus 1 1 (100%) 0%

E. coli 1 0% 0%

E. faecalis 1 1 (100%) 0%

Soft tissues 1 (2%) Zero

* 1 strain resistant to carbapenem and 2 ESBL- producing strains

**resistant to carbapenem

***ESBL producer

CNS – Coagulase-negative Staphylococcus ; MDR – Multidrug-resistant bacteria

Results 34

4.2 Surveillance cultures

A total of 4110 samples of SC were collected from three sites

(inguinal-rectal, oropharyngeal and axillary). Fifteen patients had no

samples collected by the protocol, but were not excluded because all had

had at least one SC performed for Enterobacteriaceae and CRAB during

hospital stay, which was processed in the routine microbiology laboratory.

On average, patients had SC samples every three weeks, the period

ranging between one and 14 weeks.

The site with the highest SC positivity rate for all MDR GNB was

inguinal-rectal, followed by the oropharynx. (Table 4)

The MDR-GNB SC with the lowest positivity rate was CRAB, with 19%

of patients with CRAB having negative SC; among patients with SC and

positive clinical cultures, the positivity rate of SC was higher for

carbapenem-resistant Enterobacteriaceae and lower for ESBL-producing

Enterobacteriaceae. (Table 5)

Results 35

Table 4 – Positivity rate of surveillance culture by site of harvest from the different

Multidrug-resistant Gram-negative bacteria in 180 patients submitted to liver

transplantation

MDR GNB Total number of patients

with MDR GNB

Sensitivity rate of

inguinal-rectal site

Sensitivity rate of

oropharyngeal site

Sensitivity rate of

axillary site

non-identification rate if SC was collected only from rectal site

Carbapenem resistant A. baumannii

105 53.3% 47.6% 27.6% 35.3%

Carbapenem resistant P. aeruginosa

30 80.8% 57.7% 26.9% 30.7%

Carbapenem resistant K. pneumoniae

79 92.4% 53.0% 34.8% 7.6%

Others carbapenem resistant Enterobacteriaceae

38 80.6% 52.8% 41.7% 19.4%

Enterobacteriaceae ESBL-producing

78 84.0% 36.0% 17.3% 16.0%

MDR GNB – Multidrug-resistant Gram-negative bacteria; SC – surveillance culture; ESBL – extended spectrum

betalactamases.

Table 5 – Performance of surveillance culture of different MDR GNB in 180 patients

submitted to liver transplantation

MDR GNB Total number of patients with MDR

GNB

Total number of patients

with positive SC

Positivity rate of SC among patients with positive MDR GNB clinical

culture

Proportion of patients in whom SC was the first

positive culture for MDR GNB

Carbapenem-resistant A. baumannii

105 85 (81.0%) 54 (74.0%) 59 (56.2%)

Carbapenem-resistant P. aeruginosa

30 26 (86.7%) 7 (66.7%) 26 (86.7%)

Carbapenem-resistant K. pneumoniae

79 73 (92.4%) 20 (76.9%) 70 (88.6%)

Other carbapenem-resistantt Enterobacteriaceae

38 36 (94.7%) 6 (85.7%) 5 (71.4%)

ESBL-producing Enterobacteriaceae

78 68 (87.2%) 11 (52.3%) 68 (87.2%)

MDR GNB – Multidrug-resistant Gram-negative bacteria; SC – surveillance culture; ESBL – extended spectrum

betalactamases.

Results 36

4.3 Identification of KPC-producing Enterobacteriaceae

Carbapenem-Resistant Klebsiella Pneumoniae (CRKP) was identified

in 146 samples of SC isolated from 57 patients. The blaKPC gene was

positive in 19 (33.3%) of the patients tested. The positivity of the KPC gene

among patients with CRKP identified in the 1st, 2nd, 3rd and from the 4th

week was respectively 34.8% (8/23), 26.7% (4/15), 26.7% (4/15) and 75.0%

(3/4). A tendency to increase the proportion of strains with blaKPC among

CRKP during the study period (Table 6) was observed.

Other non-K. pneumoniae CRE were identified in SC from 42 patients.

The search blaKPC gene was positive in 7 (16.7%) of the tested patients. The

distribution of positive blaKPC strains was uniform during the study period.

(Table 6)

Among clinical samples, blaKPC gene was isolated in nine patients

with positive culture by CRKP. Among patients with clinical cultures for other

carbapenem-resistant Enterobacteriaceae species, blaKPC gene was not

identified in any sample.

Table 6 – Distribution of blaKPC gene positivity rate among strains of carbapenem-

resistant Enterobacteriaceae isolated from surveillance culture

2º sem 2009

1º sem 2010

2º sem 2010

1º sem 2011

2º sem 2011

P

K. pneumoniae 0% (0/8) 20.8% (11/53) 18.2% (2/11)

20.0% (2/10)

100% (4/4)

0.02

Other Enterobacteriaceae 0% (0/5) 45.5% (5/11) 6.3% (1/16) 9.1%(1/11) 0% (0/0) 0.28

E. cloacae 0% (0/1) 50.0% (1/2) 0% (0/5) 33% (1/3)

E. aerogenes 0% (0/3) 0% (0/4) 0% (0/6) 0% (0/4)

Enterobacter spp 0% (0/1) 75.0% (3/4) 0% (0/4) 0% (0/4)

E. coli 100% (1/1) 100% (1/1)

Results 37

4.4 Analysis of risk factors for multidrug-resistant Gram-

negative bacteria acquisition after liver transplantation

Among 195 patients included in this study, 90 of them had some MDR

GNB identified in the pre-LT period, therefore the risk factor analysis for MDR

GNB acquisition after LT was performed in 105 patients. (Tables 7, 8 and 9)

After LT, 42 of the 105 (40.0%) patients acquired at least one MDR GNB.

Table 7 – Univariate analysis of risk factors for acquisition of MDR GNB after LT in 105

patients – categorical variables

Variable Patients with MDR GNB after LT (N=63)

N (%)

Patients without

MDR GNB after LT (N=42)

N (%)

P RR (CI95%)

Pre-LT characteristics

Bacterial infection before LT 13 (20.6%) 5 (11.9%) 0.25 1.11 (0.94-1.31)

Gender (F) 30 (69.8%) 13 (30.3%) 0.09 0.65 (0.39-1.09)

Hepatocellular carcinoma 15 (23.8%) 18 (42.9%) 0.04 0.75 (0.56-1.01)

Fulminant hepatitis 9 (14.3%) 1 (2.4%) 0.05 1.14 (1.02-1.27)

ASA score - - 0.79 -

LT surgical procedure characteristics

Combined liver-kidney transplant 4 (6.3%) 2 (4.2%) 1.00 1.02 (0.93-1.12)

Living-donor transplant 9 (14.3%) 9 (21.4%) 0.34 0.92 (0.76-1.11)

Senior surgeon 32 (50.8%) 21 (50.0%) 0.94 1.02 (0.69-1.51)

Duct-to-duct biliary anastomosis 60 (95.2%) 39 (92.9%) 0.68 1.50 (0.32-7.08)

Post-LT Evolution

Standard immunosuppressant therapy 29 (46.0%) 26 (61.9%) 0.11 0.71 (0.45-1.11)

Re-transplantation 10 (15.9%) 2 (4.8%) 0.12 1.13 (1.00-1.28)

Re-operation 11 (17.5%) 5 (11.9%) 0.58 1.07 (0.91-1.25)

CMV infection 3 (4.8%) 4 (9.5%) 0.43 0.95 (0.85-1.06)

Vancomycin-resistant Enterococcus colonization 30 (47.6%) 7 (16.7%) 0.01 1.59 (1.21-2.09)

Acute cellular rejection 9 (14.3%) 5 (11.9%) 0.78 1.03 (0.89-1.19)

Post-LT dialysis 28 (44.4%) 12 (28.6%) 0.10 1.29 (0.96-1.72)

MDR GNB – Multidrug-resistant Gram-negative bacteria; LT – liver transplantation; CMV - cytomegalovirus

Results 38

Table 8 – Univariate analysis for risk factors for acquisition of MDR GNB after LT in

105 patients – continuous variables

Variable

Patients with MDR GNB after LT

(N=63)

Median (min-max)

Patient without MDR GNB after LT

(N=42)

Median (min-max)

P


Pre-LT length of hospital stay (days) 1 (0-22) 1 (0-46) 0.42

Age (years) 49 (16-70) 53 (21-73) 0.31

MELD score 23 (6-53) 17 (6-34) 0.03

Pre-LT serum creatinine (mg/dl)

ASA score

1.15 (0.10-10.54) 0.94 (0.41-6.49) 0.2


Cold ischemia time (minutes) 400 (20-960) 389 (62-754) 0.42

Total ischemia time (minutes) 428 (81-910) 415 (100-730) 0.40

Duration of LT surgery (minutes) 460 (245-920) 492 (275-1330) 0.72

Units of blood transfused during LT surgery 2 (0-10) 0 (0-9) 0.02

Post-LT Evolution

Carbapenem use (days) 0 (0-32) 0 (0-30) 0.08

Quinolone use (days) 0 (0-4) 0 (0-0) 0.30

Polymyxin use (days) 0 (0-8) 0 (0-4) 0.12

Cephalosporin use (days) 2 (0-20) 2 (0-7) 0.67

Piperacillin/Tazobactan use (days) 0 (0-12) 0 (0-21) 0.98

Penicillin use (in days) 1 (0-9) 2 (0-6) 0.61

Length of dialysis after LT (days) 0 (0-24) 0 (0-50) 0.06

Length of central venous catheter (days) 6 (0-31) 4 (1-50) 0.02

Length of mechanical ventilator (days) 1 (0-17) 0 (0-9) 0.05

Length of urinary catheter (days) 6 (0-19) 4 (1-13) 0.01

Length of abdominal drain (days) 7 (0-23) 6 (0-21) 0.78

Length of ICU stay (days) 7(1-25) 4 (2-14) 0.007

MDR GNB – Multidrug-resistant Gram-negative bacteria; LT – liver transplantation; MELD – model of end-stage liver

disease, ICU – intensive care unit; ASA - American Society of Anesthesiologists (ASA) Physical Status classification.

Table 9 – Multivariate analysis of risk factors for acquisition of MDR GNB after LT in

105 patients

Variable P OR CI95%

Fulminant hepatitis 0.11 5.74 0.67-49.28

Length of urinary catheter (days) 0.02 1.16 1.03-1.32

Results 39

4.4.1 Analysis of risk factors for carbapenem-resistant K. pneumoniae

acquisition after liver transplantation

CRKP acquisition was identified in 25 patients before LT, therefore

those patients were excluded from this analysis. A total of 54 patients

acquired CRKP after LT. The analysis of risk factors for CRKP acquisition is

shown in tables 10, 11 and 12.

Table 10 – Univariate analysis of risk factors for acquisition of carbapenem-resistant K.

pneumoniae after LT in 172 patients – categorical variables

Variable Patients with carbapenem-resistant K.

pneumoniae after LT (N=54)

N (%)

Patients without

carbapenem-resistant K. pneumoniae

after LT (N=118)

N(%)

P RR (CI95%)



Gender (F) 34 (61.5%) 76 (66.1%) 0.57 0.88 (0.58-1.35)





Combined liver-kidney transplant 3 (5.6%) 6 (5.2%) >0.99 1.01 (0.93-1.09)


Senior surgeon 22 (40.7%) 61 (52.6%) 0.26 0.84 (0.62-1.13)


Post-LT evolution


Reoperation 16 (30.2%) 24 (20.7%) 0.12 1.16 (0.94-1.43)


Dialysis 32 (59.3%) 52 (44.8%) 0.08 1.65 (1.06-2.58)

Vancomycin-resistant Enterococcus

colonization 28 (53.8%) 53 (44.9%) 0.28 1.19 (0.85-1.67)


CMV infection 5 (9.3%) 15 (13.0%) 0.48 0.97 (0.86-1.08)

LT – liver transplantation; ASA - American Society of Anesthesiologists (ASA) Physical Status classification; CMV -

cytomegalovirus

Results 40

Table 11 – Univariate analysis of risk factors for acquisition of carbapenem-resistant K.

pneumoniae after LT in 172 patients – continuous variables

Variable Patients with carbapenem-resistant K.


Patients without carbapenem-resistant K.


P


Age (years) 51 (17-70) 53 (16-73) 0.35

Length of hospital stay before LT (days) 1 (0-46) 1 (0-56) 0.69

Meld score 23 (6-53) 21 (6-52) 0.78

Pre-LT serum creatinine (mg/dl) 1.20 (0.51-6.57) 1.09 (0.40-10.54) 0.49





Units of blood transfused during LT surgery 2 (0-10) 2 (0-15) 0.72

Post-LT evolution

Quinolone use (days) Zero 0 (0-6) 0.20

Piperacillin/Tazobactan use (days) 0 (0-21) 0 (0-33) 0.61


Penicillin use (days) 2 (0-9) 1 (0-21) 0.83








Length of ICU stay (days) 9 (1-72) 6 (1-71) 0.27

LT – liver transplantation; MELD – model for end-stage liver disease, CMV – cytomegalovirus

Table 12 – Multivariate analysis of risk factors for acquisition of carbapenem-resistant

K. pneumoniae after LT in 172 patients

Variable P OR CI (95%)

Dialysis after LT 0.02 2.33 1.17-4.64

Results 41

4.4.2 Analysis of risk factors analysis of carbapenem-resistant A. baumannii

acquisition after liver transplantation

CRAB was identified in 24 (12.3%) patients before LT. Among those,

four developed infection in the pre-LT period: two primary BSIs, one

pneumonia and one cholangitis. Two patients were submitted to LT with

active infection by CRAB.

The risk factors analysis for post-LT acquisition of CRAB was

performed on 171 patients.

Among the patients with CRAB, seven presented strains with reduced

MIC for polymyxin.

Tables 13, 14 and 15 show the risk factor analysis for CRAB

acquisition after LT.

Results 42

Table 13 – Univariate analysis of risk factors for acquisition of carbapenem-resistant A.

baumannii after LT in 171 patients – categorical variables

Variable Patients with carbapenem-resistant A.

baumannii after LT (N=81)

N (%)

Patients without

carbapenem-resistant A. baumannii

after LT (N=90)

N (%)

P RR (CI95%)


Bacterial infection before LT 24 (30.0) 22 (24.2) 0.39 1.08 (0.91-1.32)

Gender (F) 50 (62.5) 61 (67.0) 0.53 0.89 (0.59-1.34)

Hepatocellular carcinoma 17 (21.3) 36 (39.6) 0.01 0.77 (0.63-0.94)

Fulminant hepatitis 11 (13.8) 2 (2.2) 0.004 1.15 (1.04-1.26)

ASA score - - 0.67


Combined liver-kidney transplant 11 (13.8) 13 (14.3) 0.92 0.99 (0.88-1.12)

Living-donor transplant 4 (5.0) 5 (5.5) >0.99 0.99 (0.93-1.07)

Senior surgeon 45 (56.3) 42 (46.2) 0.19 1.23 (0.87-1.65)

Duct-to-duct biliary anastomosis 77 (96.3) 86 (94.5) 0.73 0.98 (0.92-1.05)

Post-LT evolution

Standard immunosuppressant therapy 39 (48.8) 52 (57.1) 0.27 0.84 (0.61-1.15

Reoperation 17 (20.9) 19 (21.3) 0.95 1.05 (0.86-1.17)

Retransplantation 13 (16.3) 1 (1.1) <0.001 1.19 (1.08-1.31)

Dialysis 47 (58.0) 34 (37.4) 0.005 1.52 (1.13-2.05)

Vancomycin-resistant Enterococcus colonization

9 (11.1) 17 (18.7) 0.18 0.92 (0.81-1.04)

Acute cellular rejection 44 (55.0) 30 (33.3) 0.004 2.44 (1.31 5.55)-

CMV infection 6 (7.4) 14 (15.4) 0.11 0.92 (0.82-1.02)


cytomegalovirus

Results 43

Table 14 – Univariate analysis of risk factors for acquisition of carbapenem-resistant A.

baumannii after LT in 171 patients – continuous variables

Variable

Patients with carbapenem-resistant A.


Median (max-min)

Patients without carbapenem-resistant A.


Median (max-min)

P


Pre-LT length of hospital stay (days) 1.0 (0-42) 1.0 (1.0-46.0) 0.24

Age (years) 50.0(16-70) 53.5 (18.0-73.0) 0.29

MELD score 26.0(6-51) 19.0 (6.0-5.0) 0.02



Cold ischemic time (minutes) 445.0 (20-910) 390.0 (28-754) 0.27

Total ischemic time (minutes) 461.0 (70-960) 420 (65-768) 0.23

Duration of LT surgery (minutes) 465.0 (210-920) 465.0 (245-1330) 0.48

Units of blood transfused during LT surgery 2.0 (0-15) 0.5 (0-9) 0.006

Post-LT Evolution

Carbapenem use (days) 2.5 (0-32) 0.0 (0-68) <0.001

Quinolone use (days) 0.0 (0-5) 0.0 (0-0) >0.99

Polymyxin use (days) 0.0 (0-15) 0.0 (0-26) <0.001

Cephalosporin use (days) 2.00 (0-13) 0.0 (0-20) 0.98

Piperacillin/Tazobactan use (days) 0.0 (0-16) 0.0 (0-33) 0.005

Penicillin use (days) 1.00 (0-6) 2.0 (0-16) 0.21

Length of dialysis after LT (days) 2.00 (0-58) .0.0 (0-52) 0.006

Length of central venous catheter (days) 8.0 (1.0-68.0) 4.0 (1.0-54.0) 0.01

Length of mechanical ventilator (days) 2.00 (0-25) 0.0 (0-29) 0.007

Length of urinary catheter (days) 7.0 (1.0-28.0) 5.0 (1.0-47.0) 0.08

Length of abdominal drain (days) 7.0 (0.0-83.0) 6.0 (0.0-49.0) 0.35

Length of ICU stay (days) 7.0 (1.0-28.0) 5.0 (2.0-44.0) 0.02

LT – liver transplantation; ICU – intensive care unit; MELD – model for end-stage liver disease


A. baumannii after LT in 171 patients


Hepatocellular carcinoma 0.05 0.46 0.22-0.98

Units of blood transfused during LT surgery 0.06 1.17 0.99-1.37

Re-transplantation 0.01 10.09 1.22-83.10

Previous use of carbapenem 0.001 3.31 1.66-6.60

Previous use of piperacillin / tazobactam 0.02 2.19 1.11-4.32

Results 44

4.4.3 Analysis of risk factors analysis of carbapenem-resistant P.

aeruginosa acquisition after liver transplantation

CRPA was identified in a total of 30 LT recipients. In five patients

CRPA was identified before LT, and those patients were excluded from risk

factors analysis for CRPA acquisition after LT. (Table 16, 17 and 18) No

infections by CRPA were identified in the pre-LT period.

Table 16 – Univariate analysis of risk factors for acquisition of carbapenem-resistant P.

aeruginosa after LT in 190 patients – categorical variables

Variable Patient with carbapenem-resistant P. aeruginosa

(N=25)

N (%)

Patient without

carbapenem-resistant P. aeruginosa

(N=165)

N (%)

P RR (CI95%)



Gender (F) 12 (48%) 109 (66.1%) 0.08 0.65 (0.42-1.01)

Hepatocellular carcinoma 5 (20%) 52 (31.5%) 0.24 0.86 (0.69-1.07)

Fulminant hepatitis 4 (16%) 14 (8.5%) 0.27 1.09 (0.91-1.30)



Combined liver-kidney transplant 2 (8%) 23 (13.9%) 0.54 0.94 (0.82-1.07)

Living-donor transplant 2 (8%) 10 (6.1%) 0.66 1.02 (0.90-1.15)

Senior surgeon 11 (44%) 86 (52.1%) 0.45 0.86 (0.58-1.25)

Duct-to-duct biliary anastomosis 23 (92%) 156 (94.5%) 0.61 0.68 (0.16-2.98)

Post-LT Evolution

Standard immunosuppressant therapy 12 (48%) 75 (45.5%) 0.81 1.05 (0.70-1.57)

Re-transplantation 12 (48%) 45 (27.4%) 0.04 1.40 (0.95-2.06)

Re-operation 6 (24%) 19 (11.7%) 0.09 1.16 (0.93-1.46)

CMV infection 6 (24%) 32 (19.6%) 0.61 1.06 (0.84-1.34)

Vancomycin-resistant Enterococcus colonization 17 (68%) 72 (43.6%) 0.02 1.76 (0.98-3.17)

Acute cellular rejection 6 (24%) 38 (23.2%) 0.93 1.01 (0.80-1.28)

Post-LT dialysis 20 (80%) 90 (54.5%) 0.02 2.27 (1.02-5.07)


cytomegalovirus

Results 45

Table 17 – Univariate analysis of risk factors for acquisition of carbapenem-resistant P.

aeruginosa after LT in 190 patients – continuous variables

Variable Patient with carbapenem-resistant P.

aeruginosa after LT (N=25)

Median (min-max)

Patient without carbapenem-resistant P.


Median (max-min)

P


Pre-LT length of hospital stay (days) 6 (0-31) 1 (0-67) 0.02

Age (years) 55 (25-70) 53 (16-73) 0.64

MELD score 25 (6-45) 22 (6-53) 0.08



Cold ischemic time (minutes) 460 (65-713) 390 (20-960) 0.02

Total ischemic time (minutes) 502 (105-758) 420 (65-910) 0.004



Post-LT evolution

Piperacillin / tazobactam use (days) 0 (0-16) 0 (0-33) 0.27




Penicillin use (days) 1 (0-5) 1 (0-21) 0.23






Length of ICU stay (days) 14 (0-75) 7 (1-63) 0.02

LT – liver transplantation; ICU – intensive care unit; MELD – model for end-stage liver disease


P. aeruginosa after LT in 190 patients

Variable P OR CI 95%

Length of dialysis (days) 0,12 0,97 0,94-1,01

Units of blood transfused during liver transplantation surgery 0,02 1,20 1,03-1,39

Length of intensive care unit stay (days) 0,02 1,04 1,01-1,07

Cold ischemic time (minutes) 0,07 1,01 1,00-1,01

Results 46

4.5 Healthcare-associated infections (HAI) after liver

transplantation

During the study period we identified 201 HAI in 110 (56.4%) patients

after LT.

The two most frequent sites of infection were the surgical site and BSI

(Table 19), and the two most frequent agents were A. baumannii e

K. pneumoniae. (Table 20).

Table 19 – Distribution of the sites of 201 healthcare-associated infections after liver

transplantation

Site of infection N %

Organ space surgical site 56 28%

Catheter related bloodstream 44 22%

Pneumonia 35 17%

Gastroenteritis 13 6%

Urinary tract infection 13 6%

Primary bloodstream 10 5%

Incisional surgical site 8 4%

Soft tissues 8 4%

Intra-abdominal 6 3%

Bronchitis 3 1%

Insertion catheter site 3 1%

Genital tract 1 0%

Total 201 100%

Results 47

Table 20 – Distribution of microorganism isolated from 201 healthcare-associated

infections after liver transplantation

Agent N Bacteremia (%) Multidrug-resistance N(%)

A. baumannii 56 36 (64.3%) 56 (100%)

K. pneumoniae 47 26 (55.3%) 40 (85.1%)*

E. faecium 29 15 (51.7%) 29 (100%)

P. aeruginosa 17 9 (52.9%) 13 (76.4%)

E. coli 13 7 (53.8%) 7 (53.8%)**

E. cloacae 9 4 (44.4%) 2 (22.2%)

E. aerogenes 9 4 (44.4%) 3 (33.3%)

Coagulase- negative Staphylococcous 9 5 (55.6%) 0

E. faecalis 7 3 (42.9%) 1 (14.3%)

S. aureus 6 3 (50.0%) 5 (83.3%)

M. morgannii 5 3 (60.0%) 0

C. albicans 5 1 (20.0%) -

C. glabrata 4 1 (25.0%) -

C. krusei 3 2 (66.7%) -

C. parapsilosis 2 1 (50.0%) -

P. mirabilis 2 1 (50.0%) 1 (50.0%)***

C. freundii 2 1 (50.0%) 0

A. faecalis 1 1 (100.0%) -

B. ovalus 1 1 (100.0%) -

Gram-negative bacilus 1 1 (100.0%) -

C. dubliensis 1 1 (100.0%) -

Enterococcus sp 1 1 (100.0%) 0

S. marcencens 1 1 (100.0%) 0

C. davisae 1 0 0

Aspergillus spp. 1 0 -

S. bovis 1 1 (100.0%) 0

P. stuartii 1 1 (100.0%) 0

C. pelliculosa 1 1 (100.0%) -

S. viridans 1 0 0

Total 237 132 (55.7%) 125 (52.7%)

*among the resistant isolates, 57.5% (23) were carbapenem-resistant and 42.5% (17) ESBL-producing;** ESBL-

producing; 1 strain resistant to carbapenem; *** ESBL-producing

Results 48

4.5.1 Surgical site infections

Surgical site infection corresponded to 31.8% of the HAI after LT, and

87.5% of those were classified as organ-space (OESI). In one case of

combined kidney-liver transplant, the infection was identified in the renal

graft. The time period between LT and OESI was on average 13 days, and

the median time period from LT to OESI was 10 days. In 24 (43.0%) cases

of OESI more than one agent was identified.

Among bacteria that caused OESI, 38 (77.6%) were GNB and 29

(76.3%) were classified as MDR. Two cases had no etiological agent

identified (3.6%). (Table 21)

Results 49

Table 21 – Microorganisms isolated from post-transplant organ space surgical site

infections, percentage of bacteremia and resistance profile.

Agent N Secondary bacteremia N(%)

Multidrug-resistance N(%)

A. baumannii 18 15 (83.3%) 18 (100%)

K. pneumoniae 16 9 (56%) 14 (88%)*

E. faecium 15 6 (40%) 15 (100%)

E. cloacae 6 3 (50%) 2 (33%)

E. aerogenes 5 2 (40%) 2(40%)

E. coli 4 2 (50%) 2 (50%)**

M. morgannii 4 2 (50%) 0%

P. aeruginosa 4 3 (75%) 3 (75%)

C. glabrata 3 1 (33%) -

S. aureus 3 2 (67%) 2 (67%)

S. Coagulase-negative 3 1 (33%) 0%

C. krusei 2 2 (100%) -

E. faecalis 2 0% 0%

A. faecalis 1 1 (100%) -

B. ovalus 1 1 (100%) -

Gram-negative bacillus 1 1 (100%) -

C. albicans 1 1 (100%) -

C. dubliensis 1 1 (100%) -

C. parapsilosis 1 0% -

Enterococcus sp 1 1 (100%) 0%

S. marcencens 1 1 (100%) 0%

Total Geral 94 55 (59%) -

* Among resistant isolates, 78.6% (11) were carbapenem-resistant and 21.4% (3) ESBL-producing; ** ESBL-producing

SCN – coagulase-negative Staphylococcus ; BSI – bloodstream infection

In the case of incisional surgical site infections (ISSI), an etiological

agent was identified in only half of them. No case of secondary bacteremia

was observed. The identified agents were: two coagulase-negative

Staphylococcus, one susceptible E. faecalis, one VRE and one CRAB.

Results 50

4.5.2 Bloodstream Infections after liver transplantation

Among BSI, 44 (68.7%) were classified as catheter-related BSI and

10 (18.5%) were classified as primary BSI.

In eight (15.0%) BSI more than one agent was identified. Among

isolated microorganisms, 44 (68.8%) were GNB. The most common Gram-

positive bacterium was Enterococcus in 12 (60.0%) cases. Among BSI

bacterial infections, 69% were classified as MDR, and among infections

caused by K. pneumoniae, 31.3% (5) were resistant to carbapenem. (Table 22)

Table 22 – Microorganisms isolated from post-transplant bloodstream infections and

resistance profile

Agent N Multidrug-resistance N(%)

K. pneumoniae* 16 11 (69%)

A. baumannii 14 15 (100%)

E. faecium 9 9 (100%)

E. coli** 5 3 (60%)

P. aeruginosa 4 2 (50%)

E. faecalis 3 1 (33%)

S. epidermidis 2 0%

S. haemolyticus 2 0%

C. freundii 1 0%

C. parapsilosis 1 -

C. pelliculosa 1 -

E. aerogenes 1 0%

E. cloacae 1 0%

P. mirabilis*** 1 1 (100%)

P. stuartii 1 0%

S. aureus 1 1 (100%)

S. bovis 1 0%

Total 64 -

*four strain were carbapenem-resistant and seven ESBL-producing ; ** one strain was carbapenem-resistant and two

ESBL-producing; ***ESBL-producing

The average between LT and BSI diagnosis was 28 days, and the

median was 19 days.

Results 51

4.5.3 Lower respiratory tract infection after liver transplantation

During the study period 35 pneumonia cases were identified, in one

case the causative agent was not identified, and in 13 (37.1%) cases more

than one microorganism was identified. Three cases of tracheobronchitis

were identified in the study period and none presented secondary

bacteremia. In one case, C. glabrata was implicated as the etiological agent

of empyema, this microorganism was isolated from pleural liquid with no

evidence of intra-abdominal infection. (Tabela 23)

Table 23 – Microorganisms isolated from lower respiratory tract infection, secondary

bacteremia and resistance profile.

Agent N Secondary bacteremia Multidrug-resistance N(%)

A. baumannii 20 6 (30.0%) 20 (100%)

P. aeruginosa 7 2 (29%) 7 (100%)

K. pneumoniae* 6 1 (17%) 6 (100%)

E. cloacae 2 0% 0%

E. aerogenes 2 1 (50.0%) 1 (50%)

C. glabrata 1 0% -

E. coli** 1 0% 1 (100%)

E. faecalis 1 0% 0%

C. davisae 1 0% 0%

M. morgannii 1 1 (100%) 0%

Aspergillus spp. 1 0% -

S. aureus 2 0% 2 (100%)

S. viridans 1 0% 0%

Total 47 11 (23%) -

* three strains ESBL producing and three strains resistant to carbapenem; **ESBL producing; BSI-bloodstream infection

Twenty-four cases of pneumonia (68.6%) and two (66.7%) cases of

tracheitis were associated to ventilator use.

The average length of time between LT and pneumonia was 18 days,

and the median of this interval was 15 days.

Results 52

4.5.4 Other types of infection after liver transplantation

The urinary tract infection (UTI) was the fourth most frequent infection.

No case of secondary bacteremia was identified. (Table 9) Among bacterial

infections, 86% of the agents were considered MDR. The mean time

between LT and UTI was 17 days. In two infection cases more than one

agent was identified. Four (30.8%) UTI cases were related to urinary

catheter use.

Table 24 – Microorganisms isolated from post-transplant urinary tract infections and

resistance profile.

Agent N Multidrug-resistant N(%)

K. pneumoniae* 5 5 (100%)

C. albicans 3 -

E. faecium 3 3 (100%)


C. freundii 1 0%

C. krusei 1 -

E. coli 1 0%

P. aeruginosa 1 1 (100%)

Total 16 -

*four strains were resistant to carbapenem and one was ESBL-producing

One patient had balanoposthitis by CRKP.

During the study period 13 Clostridium difficile strains were identified,

the average time between LT and Clostridium gastroenteritis was 15 days

and the median was 10 days.

Results 53

We identified eight soft tissue infections and the causative agent was

identified in only 38%; there were two cases of polymicrobial infection and

one case of secondary bacteremia by CRAB. (Table 10) 57% percent of

bacterial agents were classified as MDR.

Table 25 – Microorganisms isolated from soft tissue infections and resistance profile

Agent N Multidrug-resistant N(%)

E. coli* 2 1 (50%)


C. albicans 1 -

E. aerogenes 1 0%

E. faecium 1 1 (100%)

K. pneumoniae * 1 1 (100%)

P. aeruginosa 1 0%

Total 8 4 (50,0%)

*ESBL – producing

4.6 Analysis of risk factors for healthcare-associated

infection by MDR GNB after liver transplantation

HAI were identified in 110 (56.4%) patients after LT. Among the

patients who developed HAI, 84 (76.3%) had an MDR GNB isolated from a

clinical culture related to that infection. The analysis of risk factors for

developing HAI by MDR GNB after LT is described in Tables 26, 27, 28.

Results 54

Table 26 – Univariate analysis of risk factors for healthcare-associated infection by

MDR GNB after liver transplantation in 195 patients – categorical variables

Variable Patient with

HAI by MDR

GNB (N=84)

N (%)

Patient

without HAI

by MDR

GNB

(N=111)

N (%)

P RR (CI 95%)


Bacterial infection within 10 days before LT 32 (38.1%) 29 (26.1%) 0.07 1.19 (0.98-1.46)

Pre-LT acquisition of MDR GNB 47 (56.0%) 43 (38.7%) 0.02 1.39 (1.05-1.85)

Gender (M) 56 (66.7%) 70 (63.1%) 0.60 1.11 (0.75-1.63)





Living-donor transplant 8 (9.5%) 17(15.3%) 0.23 0.94 (0.84-1.04)

Combined kidney-liver transplant 6 (7.1%) 6 (5.4%) 0.62 1.02 (0.95-1.10)

Senior surgeon 39 (53.2%) 59 (46.4%) 0.35 0.87 (0.66-1.16)


Post-LT Evolution



Re-operation 14 (16.9%) 4 (3.6%) 0.002 1.16 (1.05-1.27)

CMV infection 11 (13.1%) 16 (14.7%) 0.75 0.98 (0.88-1.10)

Vancomycin-resistant Enterococcus

colonization

52 (61.9%) 40 (36.0%) <0.001 1.68 (1.24-2.28)

Post-LT dialysis 59 (70.2%) 44 (39.6%) <0.001 2.03 (1.41-2.91)


LT – liver transplantation, MDR GNB – Multidrug-resistant Gram-negative bacteria; HAI – healthcare-associated

infection; ASA - American Society of Anesthesiologists (ASA) Physical Status classification; CMV - cytomegalovirus

Results 55


MDR GNB after liver transplantation in 195 patients – continuous variables

Variable Patient with HAIs by MDR GNB (N=84)

Median (min-max)

Patient without HAIs by MDR GNB (N=111)

Median (min-max)

P


Pre-LT length of hospital stay (in days) 2 (0-46) 1 (0-67) 0.004

Age (years) 52 (16-70) 53 (16-73) 0.93

MELD score 24 (6-52) 19 (6-53) 0.002






Units of blood transfused during LT surgery 3 (0-15) 1 (0-9) <0.001

Post-LT Evolution




Duration of piperacilin / tazobactam use (days) 1 (0-21) 0 (0-33) 0.18

Duration of penicillin use (days) 1 (0-12) 2 (0-21) 0.23

Duration of dialysis after LT (days) 5 (0-50) 0 (0-45) <0.001

Duration of use of central venous catheter (days) 10 (1-50) 5 (1-67) <0.001

Duration of use of mechanical ventilator (days) 4 (0-30) 0 (0-10) <0.001

Duration of use of urinary catheter (days) 9 (0-35) 5 (0-35) <0.001

Duration of use of abdominal drain (days) 7 (0-60) 7 (0-34) 0.78

Duration of ICU stay (days) 10 (1-42) 5 (1-52) <0.001

LT – liver transplantation; MELD – model for end-stage liver disease, HAI – Healthcare-associated infection; MDR GNB

– Multidrug-resistant Gram-negative bacilli; ICU – intensive care unit

Table 28 – Multivariate analysis of risk factors for healthcare-associated infection by

MDR GNB after LT in 195 patients

Variable P OR CI 95%

Bacterial infection within 10 days before LT 0,33 0,68 0,31-1,48

Re-transplantation 0,04 3,85 1,06-14,03

Number of blood units transfused in LT surgery 0,005 1,25 1,07-1,46

Acquisition of MDR GNB before LT 0,05 1,98 0,99-3,93

Length of ICU stay (days) 0,04 1,05 1,02-1,11

Cold ischemia time (minutes) 0,06 1,02 1,01-1,04

LT – liver transplantation; MDR GNB – Multidrug-resistant Gram-negative bacilli; ICU – intensive care unit

Results 56

4.6.1 Analysis of risk factors analysis of healthcare-associated infection

by carbapenem-resistant K. pneumoniae after liver transplantation

A total of 23 patients developed HAI by CRKP after LT. One patient

was submitted to LT while under treatment for a CRKP active infection and

was excluded from the risk factors analysis. The risk factors analysis for

CRKP after LT is described in Tables 29, 30 e 31.


carbapenem-resistant K. pneumoniae after liver transplantation in 194 patients –

categorical variables

Variable Patients with HAIs by

carbapenem-resistant K. pneumoniae

after LT (N=23)

N (%)

Patients without HAIs

by carbapenem-resistant K. pneumoniae

after LT (N=171)

N (%)

P RR (CI95%)


Bacterial infection within 10 days before LT 10 (43.5%) 51 (29.7%) 0.18 1.25 (0.86-1.80)

Pre-LT acquisition of CRKP 7 (30.4%) 18 (10.5%) 0.007 1.29 (0.98-1.70)

Gender (M) 16 (69.6%) 110 (64.0%) 0.60 1.18 (0.62-2.27)






Combined kidney-liver transplant 2 (8.7%) 10 (5.8%) 0.64 1.03 (0.90-1.18)

Senior surgeon 14 (60.9%) 84 (48.8%) 0.28 1.31 (0.77-2.22)


Post-LT Evolution


Re-operation 7 (31.8%) 37 (21.5%) 0.28 1.15 (0.86-1.55)


Post-LT dialysis 16 (69.6%) 87 (50.6%) 0.09 1.62 (0.86-3.07)


Colonization by vancomycin-resistant Enterococcus 10 (43.5%) 82 (47.7%) 0.71 0.93 (0.63-1.36)

CMV infection 4 (17.4%) 23 (13.5%) 0.54 1.05 (0.86-1.27)

LT – liver transplantation; HAI – healthcare-associated infection; CRKP – carbapenem-resistant K. pneumoniae; ASA -

American Society of Anesthesiologists (ASA) Physical Status classification; CMV – cytomegalovirus

Results 57


carbapenem-resistant K. pneumoniae after LT in 194 patients – continuous variables

Variable Patients with HAIs by carbapenem-

resistant K. pneumoniae after LT

(N=23)

Patients without HAIs by carbapenem-

resistant K. pneumoniae after LT

(N=171)

P


Age (years) 50.0 (18.0-70.0) 53.0 (16.0-73.0) 0.81

Duration of hospital stay before LT (days) 1.0 (0.0-46.0) 1.0 (0.0-67.0) 0.15

MELD score 23.0 (7.0-52.0) 22.0 (6.0-53.0) 0.86

Serum creatinine at LT (mg/dl) 1.77 (0.57-4.26) 1.09 (0.40-10.54) 0.28


Duration of cold ischemia (minutes) 480.0 (81.0-758.0) 435.0 (65.0-960.0) 0.08

Duration of total ischemia (minutes) 445.0 (47.0-713.0) 385.0 (20.0-910.0) 0.04

Duration of LT surgery (minutes) 460.0 (250.0-850.0) 470.0 (210.0-1330.0) 0.61

Units of blood transfused during LT surgery 3.0 (0.0-12.0) 2.0 (0.0-15.0) 0.67

Post-LT Evolution

Duration of use of quinolone (days) 0.0 (0.0-4.0) 0.0 (0.0-6.0) 0.45

Duration of use of piperacilin / tazobactam (days) 2.0 (0.0-22.0) 0.0 (0.0-33.0) 0.28

Duration of use of cephalosporins (days) 1.0 (0.0-15.0) 2.0 (0.0-21.0) 0.48

Duration of use of penicillin (days) 1.0 (0.0-9.0) 1.0 (0.0-21.0) 0.25

Duration of use of polymyxin (days) 12.0 (0.0-86.0) 0.0 (0.0-77.0) 0.002

Duration of use of carbapenem (days) 20.0 (0.0-39.0) 4.0 (0.0-72.0) 0.003

Duration of use of ventilator (days) 5.0 (0.0-101.0) 1.0 (0.0-107.0) 0.06

Duration of use of abdominal drain (days) 9.0 (0.0-39.0) 7.0 (0.0-83.0) 0.49

Duration of use of central venous catheter (days) 25.0 (2.0-98.0) 8.0 (0.0-102) 0.02

Duration of use of urinary catheter (days) 15.0 (2.0-86.0) 7.0 (0.0-110.0) 0.003

Duration of use of dialysis after LT (days) 21.0 (0-91.0) 3.0 (0.0-101.0) 0.009

Duration of ICU stay (days) 18.0 (1.0-98.0) 7.0 (0.0-110.0) 0.008

LT – liver transplantation; HAI – healthcare-associated infection; MELD - model for end-stage liver disease; ICU –

intensive care unit


carbapenem-resistant K. pneumoniae after liver transplantation in 194 patients


Pre-LT acquisition of CRKP 0.003 4.70 1.72-12.85

Carbapenem use (days) 0.09 1.02 1.00-1.05

LT – liver transplantation; CRKP – carbapenem-resistant K. pneumoniae

Results 58


carbapenem-resistant A. baumannii after liver transplantation

Fifty-six cases of HAI by CRAB were identified during the study period.

Two patients had active infection by CRAB at the moment of LT and were

excluded from the risk factors analysis of infection after LT. The analysis of risk

for developing HAI by CRAB after LT is shown in Tables 32, 33 and 34.


carbapenem-resistant A. baumannii after liver transplantation in 193 patients –


Variable Patients with HAI by

carbapenem-resistant A. baumannii

after LT (N=56)

N (%)

Patients without HAI

by carbapenem-resistant A. baumannii

after LT (N=137)

N( %)

P RR (CI95%)


Bacterial infection within 10 days before LT 22 (40.0) 37 (26.8) 0.07 1.22 (0.95-2.30)

Pre-LT acquisition of CRAB 11 (19.6) 11 (8.0) 0.09 1.48 (0.95-2.30)

Gender (M) 37 (67.3) 88 (63.8) 0.65 1.11 (0.74-1.80)





Living-donor transplant 5 (9.1) 20 (14.5) 0.31 0.94 (0.84-1.04)

Combined kidney-liver transplant 3 (5.5) 8 (5.8) >0.99 0.99 (0.92-1.07)

Senior surgeon 26 (47.3) 72 (52.2) 0.54 0.91 (0.70-1.29)


Post-LT Evolution

Standard immunosuppressant therapy 25 (45.5) 81 (58.7) 0.10 0.76 (0.55-1.04)

Re-operation 16 (29.1) 35 (25.4) 0.60 1.05 (0.86-1.25)

Re-transplantation 11 (20.0) 10 (7.2) 0.01 1.16 (1.00-1.32)

Post-LT dialysis 41 (74.5) 62 (44.9) <0.001 2.16 (1.37-3.42)

Acute cellular rejection 9 (16.4) 25 (18.2) 0.77 0.98 (0.85-1.11)

Colonization by vancomycin-resistant Enterococcus 35 (63.6) 55 (39.9) 0.003 1.65 (1.14-2.41)

CMV infection 6 (10.9) 26 (19.0) 0.18 0.91 (0.80-1.02)

LT – liver transplantation; HAI – healthcare-associated infection; ASA - American Society of Anesthesiologists (ASA)

Physical Status classification; CRAB – carbapenem-resistant A. baumannii; CMV - cytomegalovirus

Results 59



continuous variables

Variable

Patients with HAI by carbapenem-

resistant A. baumannii after LT

(N=56)

Median (max-min)

Patients without HAI by carbapenem-

resistant A. baumannii after LT

(N=137)

Median (max-min)

P


Pre-LT length of hospital stay (days) 2.0 (0.0-42.0) 1.0 (0.0-56.0) 0.19

Age (years) 52.0 (16-70) 53.0 (16.0-73.0) 0.78

MELD score 27.0 (6-52) 21.0 (6.0-53.0) 0.002



Duration of cold ischemia (minutes) 441.0 (50-910) 375.0 (20.0-754.0) 0.01

Duration of total ischemia (minutes) 467.5 (95-960) 410.0 (65-798) 0.006

Duration of LT surgery (minutes) 467.5 (210.0-829.0) 460.0 (245.0-1330.0) 0.78

Units of blood transfused during LT surgery 2.0 (0.0-15.0) 1.0 (0.0-9.0) 0.004

Post-LT Evolution

Duration of use of carbapenem (days) 6.0 (0.0-40.0) 0.0 (0-72) 0.07

Duration of use of quinolone (days) 0.0 (0-0) 0.0 (0-6) 0.94

Duration of use of polymyxin (days) 0.0 (0-21) 0.0 (0-26) 0.18

Duration of use of cephalosporin (days) 2.0 (0-15) 2.0 (0-21) 0.25

Duration of use of piperacilin / tazobactam (days) 1.0 (0-19) 0.0 (0-33) 0.25

Duration of use of penicillin use (days) 0.0 (0-9) 2.0 (0-21) 0.003

Duration of dialysis after LT (days) 7.0 (0-90) 0.0 (0-78) 0.001

Duration of use of central venous catheter (days) 10.0 (1.0-49.0) 6.0 (1.0-91.0) 0.008

Duration of use of ventilator (days) 4.0 (0-30) 0.0 (0-29) <0.001

Duration of use of urinary catheter (days) 7.5 (3.0-35.0) 6.5 (3.0-56.0) 0.09

Duration of use of abdominal drain (days) 7.0 (1.0-35.0) 7.0 (0.0-49.0) 0.99

Duration of ICU stay in days 9.5 (1.0-42.0) 6.0 (2.0-44.0) 0.01

LT – liver transplantation; HAI – healthcare-associated infection; ICU – intensive care unit; MELD – model for end-stage

liver disease





Fulminant hepatitis 0.02 3.41 1.19-9.77

Pre-LT acquisition of CRAB 0.06 2.57 0.94-7.05

Cold ischemia time (minutes) 0.01 1.03 1.01-1.06

Post-LT dialysis 0.003 3.08 1.46-6.49

Carbapenem use (days) 0.25 0.98 0.96-1.01

CRAB – carbapenem-resistant A. baumannii; LT – liver transplantation;

Results 60


carbapenem-resistant P. aeruginosa after liver transplantation

No patient had an infection by CRPA before LT, and 12 developed

infection by this agent after LT. The analysis of risk factors associated to

CRPA HAI after LT is demonstrated in Tables 35, 36 and 37.


carbapenem-resistant P. aeruginosa after liver transplantation in 195 patients –



carbapenem-resistant P. aeruginosa

after LT (N=12)

N (%)

Patients without HAIs

by carbapenem-

resistant

P. aeruginosa after LT (N=183)

N (%)

P RR (CI95%)



Pre-LT acquisition of CRPA 2 (16.7) 3 (1.6) 0.03 1.18 (0.92-1.52)

Gender (M) 9 (75.0) 117 (63.9) 0.55 1.44 (0.53-3.92)


Fulminant hepatitis 0 18 (9.8) 0.61 0.90 (0.86-0.95)



Living-donor transplant 0 25 (13.7%) 0.37 0.86 (0.82-0.92)

Combined kidney-liver transplant 1 (8.3) 11 (6.0) 0.54 1.03 (0.86-1.22)

Senior surgeon 4 (33.3) 94 (51.4) 0.25 0.73 (0.48-1.12)


Post-LT Evolution


Re-operation 5 (41.7) 54 (29.5) 0.52 1.21 (0.74-1.97)

Re-transplantation 3 (25.0) 23 (12.6) 0.20 1.17 (0.84-1.62)

Post-LT dialysis 12 (100) 101 (55.2) 0.001 -

Acute cellular rejection 3 (25.0) 44 (24.2) >0.99 1.01 (0.72-1.42)


CMV infection 4 (33.3) 38 (21.0) 0.30 1.19 (0.79-1.78)

LT – liver transplantation; CRPA – carbapenem-resistant P. aeruginosa; ASA - American Society of Anesthesiologists

(ASA) Physical Status classification; CMV - cytomegalovirus

Results 61



continuous variables


carbapenem-resistant P.


Median (min-max)

Patients without HAIs by

carbapenem- resistant P.


Median (min-max)

P


Pre-LT length of hospital stay (days) 9.5 (0-31) 1.0 (0-67) 0.004

Age (years) 58.0 (25-70) 53.0 (16-73) 0.12

MELD score 23.5 (16-44) 22.0 (6-53) 0.17

Serum creatinine at LT (mg/dl) 2.14 (0.82-3.99) 1.12 (0.40-10.54) 0.08


Duration of cold ischemia (minutes) 450.5 (335-713) 385.0 (20-910) 0.02

Duration of total ischemia time (minutes) 525 (380-758) 435.0 (65-960) 0.009

Duration of LT surgery (minutes) 457.5 (277-660) 460.0 (210-1330) 0.79


Post-LT Evolution

Duration of use of piperacilin/tazobactam (days) 0.0 (0-6) 0.0 (0-33) 0.09

Duration of use of carbapenem (days) 29 (12-71) 4.0 (0-87) <0.001

Duration of use of polymyxin (days) 11.5 (3-80) 0.0 (0-81) <0.001

Duration of use of cephalosporin (days) 2.0 (0-9) 2.0 (0-21) 0.71

Duration of use of penicillin (days) 0.0 (0-6) 1.0 (0-21) 0.05

Duration of dialysis after LT (days) 27.0 (6-45) 3.0 (0-74) <0.001

Duration of use of of central venous catheter (days) 35 (8-67) 8.0 (1-92) <0.001

Duration of mechanical ventilation (days) 20.5 (3-44) 1.0 (0-78) <0.001

Duration of use of urinary catheter (days) 18.0 (5-71) 7.0 (0-98) <0.001

Duration of use of abdominal drain (days) 12.0 (2-37) 7.0 (0-83) 0.05

Duration of ICU stay (days) 28.5 (13-83) 7.0 (1-75) <0.001

LT – liver transplantation; HAI – healthcare-associated infection; MELD – model for end-stage liver disease, ICU –

intensive care unit




Variável P OR CI95%

Units of blood transfused in LT surgery 0.06 1.22 0.99-1.52

Length of ICU stay (days) <0.001 1.08 1.04-1.12

CRPA acquisition before LT 0.007 34.02 2.59-416.14

Total ischemia time (minutes) 0.08 1.005 1.00-1.01

LT – liver transplantation; CRPA – carbapenem-resistant P. aeruginosa; ICU – intensive care unit

Results 62

4.7 Evaluation of strains clonality through PFGE

We analyzed the genetic similarity between CRAB and CRKP strains

through PFGE.

Eighty-five strains of CRAB from 51 (62.2%) patients were analyzed.

Seven pulsotypes were identified during the study period: pulsotype A 5 (9.6%);

pulsotype B 34 (65.4%); pulsotype C 3 (5.8%); pulsotype D 5 (9.6%); pulsotype

E 3 (5.8%); pulsotype F 1 (1.9%); pulsotype G 1 (1.9%). In one patient, two

different pulsotypes were identified. Pulsotype B was the most prevalent and

had a homogeneous distribution during the study period. (Figure 1)

Figure 1 – Distribution of PFGE pulsotype of carbapenem-resistant A. baumannii

strains from November 2009 to November 2011

Eleven patients acquired CRAB before LT and developed infection by

this agent after LT. The paired analysis of isolates before LT and from

infection after LT was possible in 8 patients. Among those eight paired

0

5

10

15

20

25

4º trim2009

1º trim2010

2º trim2010

3º trim2010

4º trim2010

1º trim2011

2º trim2011

3º trim2011

4º trim2011

g

f

e

d

c

b

a

Results 63

isolates, five pulsotypes were identified, and in seven patients the strain

isolated after and before LT belonged to the same cluster (Figure 2)

Figure 2 – Dendogram of carbapenem-resistant A. baumannii strains from eight

patients who acquired CRAB before LT and developed infection by CRAB after LT

Twenty-six strains from 18 (75.0%) blaKPC-positive-CRKP patients

were analyzed,16 from SC and 10 from clinical cultures. Seven clusters and

nine PFGE pulsotypes were identified: cluster A 11 (42.3%); cluster B 7

(26.9%); cluster E 4 (15.4%); cluster F 1 (3.8%); cluster O 1 (3.8%); cluster

S 1 (3.8%); cluster X 1 (3.8%). Cluster A was the most prevalent one in

2010 and cluster E was more common in 2011. (Figure 3) In three blaKPC-

positive-CRKP patients it was possible to analyze the paired cultures

(before and after LT), and all strains belonged to cluster A.

Results 64

Figure 3 – Distribution of PFGE cluster of carbapenem-resistant blaKPC-positive

K. pneumoniae strains from January 2010 to November 2011

Among blaKPC-negative-CRKP strains, the analysis of genetic

similarity was performed in 36 strains from 29 (52.7%) patients. Among the

36 strains analyzed, 24 were isolated from SC and 12 from clinical cultures.

Nineteen PFGE clusters were identified: cluster C 8 (22.2%); cluster D 5

(13.9%); cluster F 1 (2.8%); cluster G 2 (5.6%); cluster H 2 (5.6%); cluster I

2 (5.6%); cluster J 3 (8.3%); cluster K 2 (5.6%); cluster L 1 (2.8%); cluster M

1 (2.8%); cluster N 1 (2.8%); cluster P 1 (2.8%); cluster Q 1 (2.8%); cluster

R 1 (2.8%); cluster T 1 (2.8%); cluster U 1 (2.8%); cluster W 1 (2.8%);

cluster Y 1 (2.8%); cluster Z 1 (2.8%). Paired isolates (before and after LT)

were analyzed in two patients, and in those two cases the strains belonged

to the same PFGE cluster. (Figure 4)

0

1

2

3

4

5

6

7

8

1º trim2010

2º trim2010

3º trim2010

4º trim2010

1º trim2011

2º trim2011

3º trim2011

4º trim2011

X

S

O

F

E

B

A

Results 65

Figure 4 – Dendogram of carbapenem-resistant K. pneumoniae isolated from Liver

transplant patients from November 2009 to November 2011

82.8

79.0

75.3

94.8

97.4

88.5

92.3

88.2

96.6

90.9

85.1

80.0

82.8

78.7

77.6

74.5

83.3

72.3

96.3

92.1

89.3

88.0

71.5

69.4

69.0

68.1

96.6

86.3

78.6

70.4

65.9

57.5

PFGE

10

0

95

90

85

80

75

70

65

60

PFGE

paciente nº LIM

18

18

12

123

118

55

60

32

33

34

44

33

55

59

34

119

68

81

62

72

81

62

180

79

167

47

56

100

21

25

29

31

39

90

131

132

115

86

115

28

94

9

9

67

11

11

50

66

76

175

183

183

183

188

147

147

146

152

3

143

160

160

Amostra

colonização pós

cultura clinica

cultura clinica

colonização pré

colonização pós

cultura clinica

cultura clinica

colonização pós

colonização pré

colonização pré

colonização pré

cultura clinica

colonização pós

colonização pós

cultura clinica

cultura clinica

cultura clinica

cultura clinica

colonização pré

colonização pós

colonização pós

cultura clinica

colonização pós

colonização pós

cultura clinica

colonização pós

colonização pós

colonização pós

colonização pós

colonização pós

colonização pré

colonização pré

colonização pós

colonização pós

colonização pós

colonização pós

colonização pré

cultura clinica

cultura clinica

colonização pré

colonização pós

cultura clinica

colonização pós

cultura clinica

colonização pré B

cultura clinica

cultura clinica

colonização pós

colonização pré

colonização pós

cultura clinica

cultura clinica

colonização pós

colonização pós

colonização pós

cultura clinica

colonização pós

colonização pós

colonização pós

cultura clinica

colonização pós

cultura clinica

KPC

neg

neg

neg

neg

neg

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

neg

pos

neg

neg

neg

neg

neg

neg

neg

neg

pos

neg

neg

neg

neg

neg

neg

pos

neg

neg

neg

neg

neg

neg

neg

neg

pos

pos

pos

pos

pos

neg

neg

neg

neg

neg

neg

neg

neg

Pulsotype

I1

I1

L

M

N

A1

A1

A1

A1

A1

A1

A1

A1

A1

A1

A2

B1

B1

B1

B1

B1

B1

B2

F1

F2

C1

C2

C3

C3

C3

C3

C3

C3

O

P

Q

J1

J1

J1

R

S

D1

D1

D2

D3

D3

T

U

W

X

E1

E1

E1

E1

K1

K2

G1

G1

Y

Z

H1

H1

Results 66

4.8 Survival analysis

During the study period, 67 (34.4%) patients evolved to death in the

first 60-days after LT. The risk factors for 60-day mortality after LT are

described in Tables 38, 39 and 40.

Table 38 – Univariate analysis of risk factors for death in the 60 first days after liver

transplantation, among 195 patients – categorical variables

Variable Patients who did not

survive within 60-days of LT (N=67) N (%)

Patients who survived

within 60-days of LT (N=128)

N (%)

P HR (IC95%)



Gender (M) 46 (68.7) 79 (62.2) 0.33 0.76 (0.46-1.30)



ASA score - - 0.05 1.54 (1.01-2.36)

Acquisition of MDR GNB before LT 35 (52.2) 54 (42.5) 0.23 1.34 (0.83-2.17)

Acquisition of carbapenem-resistant GNB before LT 26 (38.8) 32 (25.2) 0.04 1.66 (1.02-2.72)

Acquisition of CRAB before LT 12 (17.9) 11 (8.7) 0.03 1.99 (1.06-3.71)

Acquisition of CRKP before LT 12 (17.9) 12 (9.4) 0.06 1.82 (0.97-3.40)


Living-donor LT 7 (10.4) 18 (14.2) 0.43 0.73 (0.33-1.59)

Kidney-liver combined transplant 5 (7.5) 6 (4.7) 0.29 1.64 (0.66-4.07)

Senior surgeon 35 (52.2) 63 (49.6) 0.73 1.09 (0.67-1.76)

Duct-to-duct anastomosis 63 (94.0) 121 (95.3) 0.81 1.13 (0.41-3.11)

Post-LT Evolution


Reoperation 28 (41.8) 33 (26.0) 0.09 1.52 (0.94-2.48)

Re-transplantation 18 (26.9) 8 (6.3) <0.001 0.34 (0.20-0.59)

Post-LT dialysis 61 (91.0) 51 (40.2) <0.001 10.0 (4.32-23.15)

Acute cellular rejection 20 (30.3) 27 (21.6) 0.33 1.30 (0.77-2.19)


Healthcare-associated infection after LT 50 (74.6) 59 (46.5) 0.001 2.53 (1.46-4.38)

HAI by MDR bacteria after LT 45 (67.2) 42 (33.1) <0.001 2.82 (1.69-4.70)

HAI by carbapenem-resistant GNB after LT 42 (62.7) 33 (26.0) <0.001 3.12 (1.90-5.13)

HAI by CRAB after LT 32 (47.8) 24 (18.9) <0.001 2.63 (1.62-4.25)

HAI by CRKP after LT 13 (19.4) 11 (8.7) 0.06 1.78 (0.97-3.27)

Surgical site infection 26 (38.8) 27 (21.3) 0.04 1.67 (1.02-2.73)

Pneumonia 19 (28.4) 14 (11.0) 0.003 2.24 (1.31-3.81)

Bloodstream infection 20 (29.9) 28 (22.0) 0.33 1.30 (0.77-2.20)

CMV infection 9 (13.4) 35 (27.6) 0.02 0.43 (0.22-0.88)

LT – liver transplantation; MDR – Multidrug-resistant; GNB – Gram-negative bacilli; ASA - American Society of

Anesthesiologists (ASA) Physical Status classification; HAI – healthcare-associated infection; CMV - cytomegalovirus

Results 67

Table 39 – Univariate analysis of risk factors for death in the 60 first days after liver

transplantation, among 195 patients – continuous variables

Variable Patients who not survival in

60-days after LT (N=67)

Median (min-max)

Patients who survival in 60-days after LT

(N=128)

Median (min-max)

P HR (CI95%)


length of Pre-LT hospital stay (days) 2.0 (0-56) 1.0 (0-46) 0.16 1.02 (0.99-1.04)

Age (years) 55.0 (16-70) 52.0 (16-73) 0.10 1.02 (1.00-1.04)

MELD score 27.0 (6-52) 21.0 (6-53) 0.004 1.03 (1.01-1.05)

Serum creatinine at LT (mg/dl) 1.56 (0.40-6.79) 1.04 (0.44-10.54) 0.03 1.15 (1.01-1.31)


Duration of cold ischemia (minutes) 415 (20-910) 390.0 (20-740) 0.07 1.001 (1.000-1.003)

Duration of total ischemia (minutes) 460 (84-960) 420.0 (65-754) 0.05 1.002 (1.000-1.003)

Duration of LT surgery (minutes) 490.0 (250-1330) 445.0 (210-915) 0.03 1.002 (1.000-1.003)

Units of blood transfused during LT surgery 2.0 (0-15) 1.0 (0-10) 0.006 1.11 (1.03-1.20)

LT – liver transplantation, MELD – model for end-stage liver disease

Table 40 – Multivariate analysis of risk factor for death in the 60 first days after liver

transplantation, among 195 patients

Variable P HR CI 95%

Post-LT dialysis <0.001 7.91 3.33-18.94

Duration of LT surgery (minutes) 0.05 1.002 1.001-1.003

HAI by carbapenem-resistant Gram-negative bacilli 0.06 1.65 0.98-2.76

LT – liver transplantation; HAI – healthcare associated infection;

Results 68

The 60-day survival curve is shown in Figure 5.

Figure 5 – 60-day survival curve adjusted according to LT surgery duration and post-LT

dialysis, categorized by type of microorganism isolated from HAI after LT.

The P value was calculated based on group without HAI; HAI by

CRAB - <0.001; HAI by CRE – 0.06; HAI by more than one carbapenem-

resistant GNB – 0.002; HAI by non-MDR agent – 0.13.

69

5 DISCUSSION

This study was performed in a scenario of high prevalence of MDR

GNB, especially CRAB and CRKP. Although CRKP was very frequent among

LT patients in the present study, the incidence of blaKPC-producing strains

was 14.9%, which is a moderate incidence when compared to other studies

in liver transplant patients. (70) (71) Having said that, we were able to observe a

significant increase in the prevalence of blaKPC-producing-CRKP strains

during the period of this study, which was not observed for the other CRE.

The dissemination of blaKPC-producing-CRKP strains is a phenomenon

described worldwide (72) (73), therefore this epidemiological find was probably a

reflection of the general occurrence in our hospital and our city.

The incidence of CRE after liver transplantation ranges widely among

transplant centers, from 3 to 23%, and recent reports have described higher

rates of colonization and infection by CRE. (60) (71) (59) The SC for CRKP

showed a high performance, with a good sensitivity and negative predictive

value (NPV). The SC positivity rate for CRKP was highest (nearly 93%) for

samples collected from the inguinal-rectal area, a finding that is in

agreement with those of previous reports (70) (74). An important aspect

described in this study is the difference in SC sensitivity rate when CRKP is

compared with the other CRE. In the literature the sensitivity rate of CRE

Discussion 70

SC ranges between 87.5 and 100%, similar to the rate found in the present

study. (74) (70) However, the SC sensitivity rate for the other CRE in our study

was substantially lower. The comparison of SC performance between the

species of CRE is scarce in literature (74) (75) and the most plausible

explanation for this difference observed in the sensitivity rate between

CRKP and other CRE is the lowest incidence of the latter.

At our facility, CRAB was the most common MDR GNB colonization

pathogen and in early post-LT infections. Other authors have shown that

CRAB is the MDR GNB that most often colonizes LT recipients before and

after transplant. (32) Although the prevalence of CRAB colonization was quite

high in our patient sample, there are, to our knowledge, no comparative

data in literature regarding the prevalence of CRAB colonization in the

context of active, weekly surveillance after LT.

This high prevalence of MDR GNB may influence the overall rate of SC

sensitivity, which was found to be higher than that from other reports in

literature, especially for non-fermenting GNB. The sensitivity of SC for CRAB

described in literature ranges between 55 and 72%, compared to 80.6% in

the present study; (76) (77) (78) Another possible reason for the impressive

results obtained in this study is that immunocompromised patients have

higher loads of MDR microorganisms colonizing the sites sampled.

For CRAB, the perineum-rectal site proved to be the site with the best

SC positivity rate, followed by oropharynges. (77) (79) Other studies have

found that the oropharynx site had a better positivity rate than the rectal site

for CRAB isolation, however neither of these studies collected swabs from

Discussion 71

groin and rectal sites at the same time. (78) One study that analyzed different

anatomic sites of skin for CRAB found that the groin site and the perirectal

site had better sensitivity when compared to the perirectal site alone, which

allows us to infer that the groin is an important region of CRAB colonization

and therefore carrying out simultaneous SC of this region and the rectal site

may enhance the chances of identifying colonized patients. (76) On the other

hand, according to previous findings in literature and confirmed by data in

the present study, the use of only one site implicates in an important loss of

SC sensitivity for CRAB. (77)

Another important MDR GNB related to HAI in LT recipients is CRPA.

The prevalence of this agent in our service was lower when compared to

other MDR GNB. Also, when compared to other MDR GNB in this study,

CRPA had a lower proportion of colonized patients who developed infection.

Other study that evaluated living-donor LT found that the rate of pre-LT

carriers that developed infection in post-LT period was 13%; (24) In another

population this rate is described as 19.0% in HSCT recipients and 38.5% in

ICU patients. (80) (81) The SC site with the best positivity rate for CRPA was

the perineum-rectal site. Other studies that analyzed the CRAP SC positivity

rate for different sites reported that the best site was the rectal site; however

the positivity rate for this site ranged between 47% and 54%, compared to

84% in the present study. (82) (77) Among patients with CRPA isolated in

clinical cultures, 76.9% had positive SC; this finding has been described

before in HCST patients, in whom only 53.8% of the patients with CRPA

infection had CRPA detected in SC. (81) Therefore SC for CRPA seems to

Discussion 72

be an inefficient tool for direct empirical therapy, especially in the

immunocompromised population.

Our study found a low rate of infection by ESBL-producing

Enterobacteriaceae, the proportion rate of colonized patients who developed

infection was 16.3% for ESBL-producing K. pneumoniae and 16.7% for the

other ESBL-producing Enterobacteriaceae. One study that screened recipients

for ESBL-producing Enterobacteriaceae in the pre-LT period found that 48%

of LT recipients with positive SC developed infection. (83) In HSCT patients

this proportion was even higher, 61%. (84) One possible explanation for our

findings is that ESBL-producing Enterobacteriaceae are less virulent than CRE,

and so patients who were colonized by those two types of Enterobacteriaceae

had a higher probability of developing infection by CRE (85). Based on this

reasoning, we could infer that the same is applicable for CRBA and ESBL-

producing Enterobacteriaceae, since CRBA has been described as an

impressively virulent bacterium in LT recipients. (86) (35). The sensitivity of SC

for ESBL-producing Enterobacteriaceae was quite low, particularly for K.

pneumoniae. The site with the best positivity rate was the perineum-rectal

site, however if this had been the only site surveyed, more than 15% of K.

pneumoniae ESBL carriers would have been left out. Previous reports of

sensitivity for ESBL-producing Enterobacteriaceae SC range largely between

48.8 and 100%, and in most studies the only site surveyed was the rectal site,

therefore it was not possible to establish whether or not SC for ESBL-

producing Enterobacteriaceae is a good strategy for identifying the

asymptomatic carriers (87) (88) (76)

Discussion 73

Therefore this study confirms that SC was a sensitive tool in detecting

LT recipients colonized by carbapenem-resistant GNB. The SC for non-

fermenting GNB should include more than one body site. Moreover, the

active surveillance should be considered in transplant services with

moderate and high incidence of MDR GNB once the proportion of colonized

patients who developed infection is impressively high.

In the present study the only risk factor identified for acquisition

of MDR GNB was the duration of use of urinary drain counted in days.

The group of patients who acquired MDR GNB was very heterogeneous,

with different GNB species and different resistance profiles, and this may

be the major reason for the fact that few acquisition risk factors were

identified. The use of invasive devices is often described as a risk factor for

MDR bacteria acquisition in the general population and also among LT

recipients. (89) (36) (28) (72) Besides that, the use of a urinary drain is an indirect

marker of renal function impairment and also a marker of patients with less

independence in self-care activities.

In the present study, post-LT CRAB acquisition was also dependent

on the degree to which hepatic function was impaired. Patients submitted to

re-transplantation usually recover hepatic function more slowly and more

often develop post-operative complications such as biliary leaks and

thrombosis. (90) In contrast, patients submitted to LT for treatment of

hepatocellular carcinoma typically present with more preserved hepatic

function before LT, lower MELD score at LT, and fewer postoperative

complications. (91)

Discussion 74

Another major finding of the present study was that the use of broad-

spectrum antibiotics, such as carbapenem and piperacillin-tazobactam,

increased the risk for CRAB acquisition. Antibiotic use is frequently related

to the emergence of MDR bacteria and has previously been correlated with

CRAB colonization in immunocompetent individuals. (92) (93) In addition, the

use of such antibiotics before or after SOT has been associated with a

greater risk for MDR GNB infection in the recipients. (37) (18) (89)

Dialysis was found to be a risk factor for CRKP acquisition after LT in

this study. Renal failure is a well-known risk for infection after LT. (94)

Patients under dialysis usually need a central venous catheter, are more

frequently submitted to kidney-liver transplant and have a longer duration of

stay in ICU, all conditions associated with an increased risk for MDR

bacteria acquisition. (18) (89)

CRPA was, in this cohort study, the MDR GNB with the lowest

incidence. The risk factors associated to CRPA acquisition were length of

ICU stay and number of blood units transfused in LT surgery. These two

conditions may be related to an increased risk of acquiring MDR bacteria in

general in the hospital and not specifically in the LT unit. (27) (28) Patients in

ICU clearly have an increased risk of acquiring MDR bacteria, as they

usually have more invasive devices, more severe conditions and need

surgical procedures more frequently. Volume of intra-operative blood

transfusion was an indirect marker of a more complex procedure and was

previously associated to increase risk of infection after LT. (32) (95) Besides

that, transfusion itself plays an immunosuppressive role.

Discussion 75

A liver transplant surgery is a complex and prolonged procedure,

therefore surgical site infection is the most common infection reported in the

early post-LT period. (2) In the present study SSI were identified in 32% of

the patients. The majority of these infections were classified as organ-space

and more than half had secondary bacteremia associated. The data in

literature are very similar to our findings, with a reported incidence of SSI up

to 37%, the proportion of intra-abdominal commitment ranges between 67%

and 79% and bacteremia was reported in 10-46% of the cases. (96) (95) (97)

The most impressive data of our study about the epidemiology of post-

LT infection was the high incidence of GNB with a high percentage of MDR

strains. Liver transplant recipients have been described as a group of risk for

MDR bacterial infection. (98) (89) (6)The postoperative care is associated to

frequent manipulation, use of invasive devices, prolonged length of ICU stay

and frequent renal replacement therapy; also these patients receive a high

amount of broad-spectrum antibiotic in pre-transplant and early post-transplant

period. All these factors increase the risk for MDR bacteria acquisition.

As infections in early post-LT period are in their majority HAI,

the microbial agent profile is closely related to the overall epidemiology of

the institution where transplantation occurs. (2) The most recent studies

report a tendency of an increase in MDR GNB as agents of post-LT early

infections. (98) (89) Bert et al in a cohort study of 704 LT recipients reported

that the most frequent agents of BSI in the first year after LT were

Enterobacteriaceae species. (30) Shi et all in a cohort study of 475 LT

recipients described that 32% developed bacteremia due to GNB in the first

Discussion 76

six months after LT, and 56.3% of those GNB isolated were categorized as

MDR; the most frequent MDR GNB was CRAB. (18) In contrast a Spanish

study that analyzed all bacteremia cases occurring in hospitalized SOT

recipients during a five-year period, found that CRPA was the most frequent

MDR bacteria in LT recipients. (6)

The CRPA is a common agent related to HAI after LT; the most

common site of CRPA infection is pneumonia, CRPA being isolated in up to

18% of this type of infection. (31) (99) (24) (100) CRPA can be the causative agent

in 9% of BSI. (6) (89) In this cohort study CRPA infection was identified in 6.2%

of patients after LT, and the most common site was also pneumonia and was

the agent isolated in 18.4% of the infections in the lower respiratory tract.

Risk factors identified in the present study associated to MDR GNB

infection were: prolonged ICU stay, large amount of blood transfusion during

LT surgery, re-transplantation and colonization by MDR GNB prior to LT.

The amount of blood transfusion during LT surgery and re-

transplantation were two variables associated with graft complication. It was

expected that variables associated with an increased risk for SSI would be

identified in the risk factor analysis for MDR GNB infection, once SSI

represented a great number of infections in our cohort study and as

described in literature infections by MDR bacteria were more commonly

isolated from graft infection than from SSI. (71) (35) (89)

As discussed for CRAB acquisition, re-transplantation is associated

to slower recovery of hepatic function and more frequent postoperative

Discussion 77

complications such as biliary leaks and thrombosis, which also increases

the risk for post-LT infection. (90) A Spanish cohort study of 276 episodes of

bacteremia in SOT recipients described that patients with prior

transplantation had an increased risk for current bacteremia by MDR

bacteria. (6) A study designed to identify risk for SSI after LT also found that

re-transplantation was associated with higher number of SSI. (96)

Amount of blood transfused during LT surgery, as discussed earlier,

is a marker of complication during this procedure. In literature patients who

received more blood during transplant surgery had an increased risk

of developing SSI; this finding was described in LT but also in other types

of SOT. (101)

Patients in ICU have an increased risk for developing infection, due to

the severity of the patient and the degree of invasiveness they are

submitted to. In other populations ICU stay was frequently associated with

risk for MDR GNB infection, such CRKP and CRAB. (85) (28) One study that

evaluated bacteremia by P. aeruginosa in transplant patients identified that

previous ICU stay was a risk for bacteremia by CRPA in non-lung transplant

recipients. (31)

The incidence of post-LT CRE infection also varies among transplant

centers, ranging from 3% to 23%, and recent studies have reported higher

rates of CRE colonization and infection. (71) (60) (59) At our facility, the

incidence of CRE infection is high, as evidenced by the number of colonized

patients in the present study, as well as by the progressive increase in the

proportion of KPC-producing strains over the study period.

Discussion 78

An interesting aspect described in this study is that despite the fact

that the rate of colonized patients who developed infection in a relatively

short period of follow-up was high (27.3%), this proportion is lower than was

described in other reports of LT recipients, which ranges between 39.0%

and 88.9%. (70) (59) One possible explanation for that finding is that blaKPC-

producing CRKP are described as more virulent than the strains that harbor

other types of carbapenem resistance mechanisms and in this cohort the

majority of CRE were blaKPC negative. (85)

There have been few studies analyzing the risk of CRE acquisition

and infection in Liver transplant patients in a context of weekly screening.

Giannella et al. analyzed 237 LT recipients who were screened weekly for

CRE starting at LT and they identified CRE acquisition at any time (pre- or

post-LT) as the variable most strongly correlated with post-LT CRE infection.

(59) In our study, pre-LT acquisition was also the only risk factor identified for

developing CRE infection.

The reported prevalence of post-transplant CRAB infection among

LT recipients varies widely across facilities, ranging between 2.8% and

16.8%. (6) (18) (102) In the present study, the prevalence of CRAB infection was

high, 27.7%. In our analysis, we identified other factors associated with an

increased risk of post-LT CRAB infection: post-LT need for dialysis,

prolonged cold ischemia time, and LT performed due to fulminant hepatitis.

Cold ischemia time longer than 400 minutes has previously been identified

as a risk factor for SSI after LT, (32) and in the present study a prolonged

total ischemia time was associated with an increased risk for infection by

Discussion 79

CRPA. In general, it can be assumed that longer ischemia time is

associated with slower post-LT recovery of the systemic inflammatory

response, as well as, indirectly, longer ICU stays, increased use of invasive

devices, and delayed recovery of renal function. (103) (104)

The need for dialysis after LT is a well-known risk factor for early

postoperative bacterial infections. (94) Patients requiring dialysis are more

often subjected to the use of invasive devices and are more often admitted

into ICU, thus having a higher incidence of bacterial infection. (89) (18) Renal

failure itself plays an immunosuppressive role and can be an indirect marker

of graft malfunction. Acute kidney injury can also be seen in LT recipients

with severe preoperative hepatic impairment. Gouvêa et al. reported that

hemodialysis after SOT is more common among recipients who develop

infection with CRAB than among those who develop infection with

carbapenem-susceptible A. baumannii. (37)

Patients undergoing LT due to fulminant hepatitis typically show high

MELD scores at the time of transplantation, and such patients also often

have longer ICU stays and need dialysis before LT. (32) A high MELD score

at LT has been associated with a greater risk of bacterial infection,

specifically post-LT bacteremia caused by Enterobacteriaceae. (105) In a

univariate analysis, Hsieh et al. demonstrated that a high MELD score is a

risk factor for A. baumannii infection after LT. (33)

This study has demonstrated that the acquisition of MDR GNB in pre-

LT can increase the risk for developing infection by these agents in post-LT

period. This was also true when specific MDR GNB were separately

Discussion 80

analyzed, namely CRKP, CRAB and CRPA. One possible explanation for

this finding is that pre-LT MDR GNB acquisition could be a marker of poorer

clinical conditions, which would increase the risk of developing post-LT

complications. Another possibility is that the standard antibiotic prophylaxis

might have been less effective in the patients colonized by MDR GNB.

There is little knowledge regarding outcomes in SOT recipients

colonized by MDR GNB before transplant, and the impact of modifying the

surgical prophylaxis has not been established. (99) (101) In the present study,

modifying the surgical prophylaxis did not prevent the occurrence of post-LT

GNB MDR infection. However, because of the small number of cases in

which the prophylaxis was adjusted, it was not possible to draw a conclusion

about the efficacy of that measure. At our facility, surgical prophylaxis is not

systematically modified on the basis of colonization of the recipient by MDR

bacteria, but rather only on the basis of preoperative infection. It is therefore

likely that the patients in whom the surgical prophylaxis was modified were

those who were the sickest. In addition, the total number of patients

recognized as colonized before LT was too small to demonstrate the benefit

of this strategy.

In addition, the PFGE analysis showed that most of the patients

developed post-LT infection from the same strain that they harbored in the

pre-LT period. That was also true for the patients who harbored strains

other than the predominant clone. The impact that pre-LT colonization by

MDR GNB has on infectious outcomes after LT has not been well defined.

Kim et al. reported that, in a small number of LT recipients, colonization by

Discussion 81

A. baumannii, identified in sputum on the day of LT, was associated with the

risk of A. baumannii-associated pneumonia in the post-transplant period. (99)

In another study, involving 475 LT recipients, microbiologically proven pre-

LT infection was found to be a risk factor for post-LT death from MDR GNB

infection. (18)

In one study, surveillance samples were collected to culture for

extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae

before LT (19). The authors also found that pre-LT acquisition substantially

increased the risk of infection in the post-LT period; 48% of the patients

colonized in the pre-LT period developed infection with ESBL-producing

Enterobacteriaceae after LT, compared with 6.7% of the patients not

colonized (19).

One study in living-donor liver transplant analyzed the impact of pre-LT

colonization by CRPA in risk of infection after LT by this agent. This study did

not find any correlation, however there was no active screening for detection

of CRPA colonization in any time during pre and post LT period. (24)

We performed an analysis for 60-day mortality risk factors in order to

investigate the role of MDR GNB infection in LT recipient surveillance. The

infection by carbapenem-resistant GNB showed a considerable trend

toward significance as risk for 60-day mortality.

In our study infection by carbapenem-resistant GNB after LT was

related to increased 60-day mortality after LT. Although the p-value showed

a trend toward significance, this finding has a high biological plausibility.

Discussion 82

Other studies that analyzed infection by MDR GNB in LT recipients usually

found a high associated mortality. (70) (71) (35) (3) In the survival analysis,

infection by CRGNB infection had the best fitness in the final model

compared to other MDR GNB infections. In spite of that, in survival plot it is

possible to observe that CRAB had the highest mortality rate when

compared to patients with other MDR GNB infections.

Post-SOT infection with A. baumannii is associated with high mortality,

reportedly as high as 80.0%, and carbapenem resistance appears to increase

the risk of death significantly. In literature, the mortality associated with CRE

infection in LT recipients’ ranges between 41% and 72%. (59) (70) (56) (71) (106)

Because SOT recipients infected with MDR bacteria are more likely to

receive initial antibiotic therapy that is inappropriate, to have longer ICU stay,

and to require mechanical ventilation, mortality rates are higher among such

patients than among SOT recipients infected with bacteria that are drug-

susceptible and among those without infection. (6) (102) (98)

Other risk factors identified in our 60-day risk factor analysis were

to have performed dialysis after LT and prolonged LT surgical time.

Renal impairment was consistently associated to a lower survival in post-LT.

A national wide study in Finland that included 631 LT recipients found that

glomerular filtration rate is inversely proportional to patient survival. (107)

Another large population based cohort study from Taiwan that included

2938 patients submitted to LT described that dialysis increase the mortality

rate in up to 100%. (108)

Discussion 83

Surgical time length is a very good marker for surgery complexity.

Longer surgical times were associated to increased mortality once they

have a worse systemic inflammatory response syndrome, increase in the

risk of infection, and are more associated to technical surgeries

complication as bleeding and biliary lesions. (109) (110)

In conclusion, we found that patients who acquired MDR GNB prior to

LT were at a greater risk for post-LT MDR GNB infection, which has a

negative impact on recipient’s survival. We also found that the risk of MDR

GNB infection was higher among patients submitted to re-transplantation,

patients who received large amount of blood units during LT surgery and

had a prolonged ICU stay. Patients who were colonized by MDR GNB when

they underwent LT should be carefully evaluated for the best moment for LT,

and measures for minimizing that risk, such as adjusting the antibiotic

prophylaxis or decolonization treatment, should be investigated and

implemented.

84

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7 APPENDIX

Manuscripts derived from this thesis published in index medical

journals:

A. Surveillance culture for multidrug-resistant gram-negative bacteria: Performance in liver transplant recipients. Freire MP, Villela Soares Oshiro IC, Bonazzi PR, Pierrotti LC, de Oliveira LM, Machado AS, Van Der Heijdenn IM, Rossi F, Costa SF, Carneiro D'Albuquerque LA, Abdala E. Am J Infect Control. 2017 Mar 1;45(3):e40-e44. doi: 10.1016/j.ajic.2016.12.010.

B. Carbapenem-Resistant Enterobacteriaceae Acquired Before Liver Transplantation: Impact on Recipient Outcomes. Freire MP, Oshiro IC, Pierrotti LC, Bonazzi PR, de Oliveira LM, Song AT, Camargo CH, van der Heijden IM, Rossi F, Costa SF, DʼAlbuquerque LA, Abdala E. Transplantation. 2017 Apr;101(4):811-820. doi: 10.1097/TP.0000000000001620

C. Carbapenem-resistant Acinetobacter baumannii acquired before liver transplantation: Impact on recipient outcomes. Freire MP, Pierrotti LC, Oshiro IC, Bonazzi PR, Oliveira LM, Machado AS, Van Der Heijden IM, Rossi F, Costa SF, D'Albuquerque LA, Abdala E. Liver Transpl. 2016 May;22(5):615-26. doi: 10.1002/lt.24389

D. Polymyxin use as a risk factor for colonization or infection with polymyxin-resistant Acinetobacter baumannii after liver transplantation. Freire MP, Van Der Heijden IM, do Prado GV, Cavalcante LS, Boszczowski I, Bonazzi PR, Rossi F, Guimarães T, D'Albuquerque LA, Costa SF, Abdala E. Transpl Infect Dis. 2014 Jun;16(3):369-78. doi: 10.1111/tid.12210. Epub 2014 Apr 12.

https://www.ncbi.nlm.nih.gov/pubmed/28254253








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Documents

Aquisição de bactéria gram-negativa multidroga resistente ... · This thesis is in accordance with the following rules, validity at the moment of the publication. References: adapted